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WO2019126364A2 - Compositions and methods related to nicotine addiction and cessation - Google Patents

Compositions and methods related to nicotine addiction and cessation Download PDF

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Publication number
WO2019126364A2
WO2019126364A2 PCT/US2018/066562 US2018066562W WO2019126364A2 WO 2019126364 A2 WO2019126364 A2 WO 2019126364A2 US 2018066562 W US2018066562 W US 2018066562W WO 2019126364 A2 WO2019126364 A2 WO 2019126364A2
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Prior art keywords
nicotine
nica2
degrading
enzyme
fusion protein
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French (fr)
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WO2019126364A3 (en
Inventor
Kim D. Janda
Olivier GEORGE
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Scripps Research Institute
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01328—Nicotine blue oxidoreductase (1.1.1.328)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to methods and compositions for promoting nicotine cessation and for treating nicotine addiction.
  • the invention provides novel nicotine-degrading fusion proteins that can degrade nicotine in vivo and treat disorders related to nicotine consumption and addiction.
  • the nicotine-degrading fusion proteins of the invention contain aN-terminal truncated nicotine-degrading enzyme and an albumin binding moiety.
  • the nicotine-degrading fusion enzyme is NicA2 isolated from Pseudomonas putida S16 or a conservatively substituted variant thereof.
  • the albumin binding moiety is albumin binding domain (ABD)035 or a conservatively substituted variant thereof.
  • the employed N-terminally truncated nicotine degrading enzyme is a NicA2 variant with a deletion of about 25 or more N-terminal amino acid residues of the wildtype enzyme. In some of these embodiments, the deletion is about 45, 46, 47, 48, 49, 50, 51 or more N-terminal amino acid residues.
  • the albumin binding moiety is fused to the N-terminus of the truncated enzyme.
  • Some nicotine-degrading fusion proteins of the invention contain aNicA2 variant with a deletion of about the first 50 N- terminal residues, and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
  • the fusion enzyme has substantially the same or better substrate binding and/or catalytic activity relative to the wildtype NicA2 enzyme.
  • the truncated enzyme is linked to the albumin binding moiety via a linker moiety.
  • the invention provides polynucleotide sequences encoding a nicotine-degrading fusion protein that contains a N-terminal truncated nicotine-degrading fusion enzyme and an albumin binding moiety.
  • the encoded nicotine degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
  • the invention provides methods of treating nicotine addiction and/or promoting nicotine cessation in a human patient suffering from nicotine addiction and/or nicotine consumption. These methods entail administering to the patient in need of treatment a therapeutically effective amount of a nicotine-degrading fusion protein described herein.
  • the employed nicotine-degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
  • the effective amount of the administered enzyme is an amount that is sufficient to reduce compulsive-like behavior and/or irritability -like behavior in the patient.
  • the invention provides methods for preventing relapse of nicotine dependence in a human patient exhibiting symptoms of nicotine dependence. These methods involve administering to the patient a therapeutically effective amount of a nicotine degrading fusion protein described herein.
  • the administered nicotine degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
  • the effective amount of the administered enzyme is an amount that is sufficient to reduce compulsive-like behavior and/or irritability -like behavior in the patient.
  • the invention provides methods for treating nicotine poisoning in a human patient in need thereof.
  • the methods involve administering to the patient a therapeutically effective amount of a nicotine-degrading fusion protein described herein.
  • the administered nicotine-degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
  • the effective amount of the administered enzyme is an amount that is sufficient to ameliorate one or more symptoms associated with nicotine poisoning.
  • Figure 1 shows pharmacokinetics of NicA2 and NicA2-Jl in rat serum.
  • FIG. 1 Figure shows effect of NicA2-Jl during nicotine withdrawal in rats.
  • NicA2-Jl blocks total withdrawal score (A), hyperalgesia (B) irritability like behavior (C) and during withdrawal.
  • Figure 3 shows nicotine concentrations in rat blood (A) after 1 or 5 days and brains (B) after 7 days of NicA2-Jl administration. (ND: not detected).
  • Figure 4 shows blood nicotine levels after escalation of nicotine intake. Detailed timeline of the experiments (upper panel).
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 1 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal.
  • FIG. 5 shows that NicA2-Jl prevents nicotine-
  • FIG. 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia.
  • FIG. 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia.
  • FIG. 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia.
  • FIG. 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia.
  • FIG. 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia.
  • Figure 7 shows effect of NicA2-Jl on the escalation of nicotine intake (1 and 21 h).
  • Top Detailed timeline of the experiments (upper panel).
  • FIG. 8 shows that NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats.
  • FIG. 8 shows that NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats.
  • FIG. 8 shows that NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats.
  • FIG. 8 shows that NicoA2-Jl reduces compulsive-like responding for nicotine in dependent rats.
  • FIG. 8 shows that NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats.
  • FIG. 9 shows that NicA2-Jl prevents nicotine- and stress-induced reinstatement after extinguished nicotine intake.
  • the present invention is predicated in part on the studies undertaken by the inventors to develop nicotine-degrading enzymes suitable for clinical uses in human subjects.
  • the inventors explored an alternative strategy to reducing nicotine’s brain concentration.
  • this alternative strategy ensures that an effective concentration could not be reached or maintained in the brain.
  • the nicotine-degrading enzymes developed by the inventors can reverse nicotine dependence, decrease compulsive- like intake, and prevent relapse in a translational animal model of nicotine addiction.
  • the enzymes e.g., the NicA2-Jl fusion protein exemplified herein
  • the enzymes have a favorable pharmacokinetic profile, including a relatively long half-life and simple route of administration.
  • a fusion protein based on a truncated nicotine degrading enzyme NicA2 and albumin binding domain (ABD)035, which demonstrated significantly improved in vivo stability.
  • the exemplified fusion protein, NicA2-Jl has demonstrated surprisingly advantageous properties relative to NicA2 or variants that are known in the art.
  • NicA2-Jl was found to have catalytic activity that is similar to the wildtype enzyme NicA2 but substantially improved serum stability.
  • the fusion enzyme is able to degrade nicotine and prevents the development of nicotine dependence in rats. Serum nicotine distribution and behavioral testing revealed that the fusion enzyme completely eliminates blood nicotine content, thereby halting the drug’s psychoactive effects.
  • the inventors also conducted a series of studies to determine whether NicA2-Jl has translational relevance and prevents addiction-like behaviors in animals with a history of compulsive-like nicotine self-administration. It was demonstrated that NicA2-Jl decreased blood nicotine levels and had remarkable preclinical efficacy in reducing addiction-like behaviors in nicotine-dependent rats. NicoA2-Jl administration reduced blood nicotine levels, reversed somatic and emotional signs of nicotine withdrawal in dependent rats, reduced compulsive-like responding for nicotine, and prevented nicotine- and stress-induced relapse.
  • Nicotine-dependent rats that were treated with NicA2-Jl exhibited a lack of hyperalgesia and a robust decrease in irritability -like behavior after 2 weeks of treatment.
  • acute NicA2-Jl treatment did not precipitate withdrawal during nicotine self-administration and decreased withdrawal-induced hyperalgesia when administered acutely.
  • NicA2-Jl prevented the development of nicotine dependence and reversed nicotine dependence by normalizing somatic and emotional signs of nicotine withdrawal in only 2 weeks. This is a critical result because irritability during abstinence is often mentioned by users as one of the primary reasons why they relapse. Even more compelling from a translational perspective, NicA2-JI decreased compulsive-like responding for nicotine, reflected by nicotine intake despite the adverse consequences of contingent footshocks. This suggests that treatment with the NicA2-Jl enzyme reduced symptoms of nicotine withdrawal and diminished the incentive value of nicotine, thereby decreasing the motivation to take nicotine when confronted with adverse consequences. NicoA2-Jl did not affect nicotine self-administration when no adverse consequences were presented, suggesting that the very low blood nicotine levels were sufficient to serve as a discriminative stimulus but not sufficient to have incentive value, including responding despite adverse
  • NicA2-Jl reduced compulsive-like nicotine seeking, which is highly relevant to the human condition, in which smoking is often associated with significant adverse social consequences (e.g., conflicts with partners) and health consequences (e.g., coughing, pulmonary disease, and cancer) that are often ignored by smokers because of the high incentive value of nicotine and the ability of nicotine to provide relief from withdrawal symptoms.
  • adverse social consequences e.g., conflicts with partners
  • health consequences e.g., coughing, pulmonary disease, and cancer
  • the invention provides nicotine degrading fusion enzymes that are comprised of aN-terminally truncated nicotine degrading enzyme and a fusion partner that enhances the stability and in vivo half-life of the enzyme while maintaining its enzymatic activities.
  • the invention also provides polynucleotide sequences that encode the fusion enzymes described herein, as well as vectors harboring such that harbor the polynucleotide sequences for expressing the fusion enzymes.
  • the invention further provides methods of using the fusion enzymes, the encoding polynucleotides and expression vectors in various therapeutic and prophylactic applications for countering nicotine use related medical conditions and symptoms.
  • the various methods described herein are directed to treating nicotine addiction, treating nicotine-addiction related disorders, reducing the risk of relapse of nicotine consumption, promoting smoking cessation, extending a duration of smoking abstinence in a subject who has quit smoking, increasing a likelihood of long-term abstinence from smoking, and/or rescuing a subject from relapse of nicotine consumption.
  • the nicotine degrading fusion enzymes of the invention can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; lst edition (1997) (ISBN-13: 978- 0121821906); U.S. Pat. Nos.
  • compositions and methods of the present invention are provided.
  • a nicotine degrading fusion enzyme can refer to both single or plural nicotine degrading fusion enzymes, and can be considered equivalent to the phrase “at least one nicotine degrading fusion enzyme.”
  • conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a fusion protein is a protein molecule containing amino acid sequence from at least two unrelated proteins that have been joined together, e.g., via a peptide bond, to make a single protein.
  • the unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence.
  • proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell).
  • Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443,
  • subject refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms“patient” or“subject” are used herein interchangeably. Preferably, the subject is human.
  • the term“treating” or“alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease or condition (e.g., nicotine addiction or withdrawal symptom), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder.
  • Treatment may be prophylactic (to prevent or delay the onset of the disease or condition, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.
  • the invention provides nicotine degrading fusion enzymes and clinical applications of the same.
  • the nicotine degrading fusion enzymes of the invention are comprised of aN-terminally truncated nicotine-degrading enzyme and a fusion partner for enhanced stability and in vivo half-life.
  • the employed nicotine degrading enzyme for truncation is obtained from bacterium Pseudomonas memeida.
  • the nicotine-degrading enzyme for truncation is NicA2.
  • NicA2 was originally isolated from Pseudomonas putida (Strain 16). See, e.g., Tang et al, PLoS Genet.
  • the variant NicA2 enzyme used in the fusion protein of the invention typically has a N-terminal truncation relative to the wildtype enzyme.
  • the truncation comprises a deletion of about the first 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more of the N-terminal amino acid residues.
  • the truncation constitutes a deletion of the first 50 N-terminal residues of the wildtype NicA2 (i.e., D50- NicA2).
  • the truncation constitutes a deletion of the first 41, 42, 43, 44, 45, 46, 47, 48, or 49 N-terminal residues of the wildtype NicA2.
  • the truncation constitutes a deletion of the first 51, 52, 53, 54 or 55 N-terminal residues of the wildtype NicA2 enzyme.
  • the employed variant enzyme can have one or more of the amino acid residues that are different from what is present in the wildtype nicotine-degrading enzyme.
  • a variant nicotine-degrading enzyme has at least 75%, 80%, 85%, 90% or 95% sequence identity with the amino acid sequence of the wildtype nicotine-degrading enzyme, such as NicA2.
  • the NicA2 variant enzyme has at least 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 1. in some embodiments, the NicA2 variant enzyme has at least 96%, 97%, 98% or at least 99% sequence identity with the wildtype NicA2 enzyme.
  • the employed NicA2 variant enzyme contains one or more conservatively substituted amino acid residues relative to the wildtype enzyme.
  • the variant nicotine-degrading enzyme used in constructing the fusion protein of the invention e.g., a conservatively substituted variant of A50-NicA2 should maintain substantially the same enzymatic function or in vivo nicotine degrading activity of the wildtype protein.
  • the employed variant should have substantially the same or better substrate binding and catalytic activity relative to the wildtype NicA2 enzyme or the ⁇ 50-NicA2 variant exemplified herein.
  • enzy matic function of the vanant enzymes e.g., k cat and K m values
  • k cat and K m values can be readily determined via any of the in vitro or in vivo assay s exemplified herein or that is well known in the art. See, e.g., WO2017023904.
  • the fusion enzyme of the invention contains the N-terminal truncated nicotine degrading enzyme (e.g., ⁇ 50-NicA2 exemplified herein) that is linked to a fusion partner.
  • N-terminal truncated nicotine degrading enzyme e.g., ⁇ 50-NicA2 exemplified herein
  • the fusion partner can be any moiety that increases the circulating half-life of the truncated nicotine-degrading enzyme in vivo, e.g., an albumin-binding moiety, an albumin moiety or a polyethylene glycol moiety.
  • the employed fusion partner is an albumin-binding moiety.
  • some fusion enzymes of the invention contain the N-terminal truncated NicA2 that is fused to albumin-binding domain ABD(035).
  • ABD(035) is an albumin binding protein domain that is well known in the art. See, e.g., Jonsson et al., Protein Eng. Des. Sel. 2l(8):515-27, 2008.
  • the fusion partner can be conjugated or linked to the truncated enzyme at either the N-terminus or the C-terminus of the enzyme.
  • the fusion partner e.g., ABD(035), is fused to the N- terminus of the enzyme.
  • the method for generating the fusion protein of the invention is not subject to any particular limitation.
  • the fusion protein of the invention may be a fusion protein synthesized by chemical synthesis, or a recombinant fusion protein produced by a genetic engineering technique. If the fusion protein of the invention is to be chemically synthesized, synthesis may be carried out by, for example, the Fmoc (fluorenylmethyloxy carbonyl) process or the tBoc (t-butyloxy carbonyl) process.
  • peptide synthesizers available from, for example, Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Syntheceh-Vega, PerSeptive and Shimadzu Corporation may be used for chemical synthesis.
  • the fusion proteins of the invention are produced by genetic engineering using the conventional recombination techniques routinely practiced in the art. Such techniques are described, e.g., in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
  • Recombinant production of the fusion enzymes of the invention typically involves removing the stop codon from a polynucleotide sequence (e.g., a cDNA sequence) coding for the fusion partner (e.g., an albumin binding protein), then appending a polynucleotide sequence (e.g., a cDNA sequence) encoding the truncated nicotine-degrading enzyme (e.g., ⁇ 50-NicA2) in frame through ligation or overlap extension PCR.
  • the fusion protein can then be expressed by inserting the resulting polynucleotide sequence encoding the fusion protein into a suitable expression system.
  • linker moiety or spacer peptide may be used for linking the two components of the fusion enzymes.
  • linker moieties e.g., GS linker or G 4 S linker
  • the fusion enzyme proteins of the invention may additionally include a peptide sequence for purification.
  • Peptide sequences for purification that may be used are also known in the art.
  • Examples of peptide sequences for purification include histidine tag sequences having an amino acid sequence in which at least four, and preferably at least six, continuous histidine residues, and the amino acid sequence of the glutathione-binding domain in glutathione S- transferase.
  • the N-terminally truncated NicA2 fusion enzymes of the invention are generated by recombinantly linking an albumin binding protein domain to the N-terminus of the truncated NicA2 enzyme.
  • Some specific exemplifications are discussed in detail in the Examples below.
  • construction of the fusion enzyme containing the N-truncated nicotine-degrading enzyme (e.g., ⁇ 50-NicA2) to a fusion protein partner (e.g., ABD(035)) can be readily carried via the methods exemplified herein or standard protocols of biochemistry and molecular biology.
  • Cloning and recombinant expression of truncated NicA2 and the albumin binding protein domain can be performed with techniques exemplified herein. Fusion of the enzyme to the albumin binding domain can be similarly carried out as exemplified herein for the generation of the NicA2-Jl fusion enzyme which contains the ABD(035) domain fused to the N-terminus of ⁇ 50-NicA2.
  • related embodiments of the invention include polynucleotide sequences that encode such fusions, expression constructs for expressing the fusion proteins, and host cells that harbor the polynucleotides or expression constructs.
  • the polynucleotide sequences of the invention can be any polynucleotide having a nucleotide sequence that encodes the fusion protein of the invention, although DNA is preferred.
  • the recombinant constructs or expression vectors of the invention harbor a polynucleotide sequence of the invention that encodes a N-terminally truncated nicotine-degrading fusion enzyme.
  • the recombinant constructs of the invention may be obtained by ligating (inserting) the polynucleotide (DNA) of the invention into a suitable vector. More specifically, the recombinant vector may be obtained by cleaving purified polynucleotide (DNA) with a suitable restriction enzyme, then inserting the cleaved polynucleotide to a restriction enzyme site or multicloning site on a suitable vector, and ligating the polynucleotide to the vector.
  • the vector for inserting the polynucleotide sequence is not subject to any particular limitation, provided it is capable of replication in an appropriate host.
  • the expression vectors can be, for example, bacteriophages, plasmids, cosmids or phagemids.
  • recombinant bacteriophage or phagemid vectors include that based on a filamentous phage such as Ml 3.
  • Plasmid vectors include those based on plasmids from, e.g., E. coli (e.g., pBR322, pBR325, pUCH8 and pUCH9), plasmids from Bacillus subtilis (e.g., pUBl lO and pTP5), and plasmids from yeasts (e.g., YEpl3, YEp24 and YCp50).
  • the expression vectors can also include vectors derived from animal viruses such as retroviruses, vaccinia viruses and insect viruses (e.g., baculoviruses).
  • the invention also provides therapeutic and prophylactic methods for treating nicotine toxicity, nicotine addiction, promoting smoking cessation, reducing the relapse of nicotine consumption, preventing or reducing symptoms associated with nicotine withdrawal, or treating nicotine poisoning.
  • treatment via methods of the invention is intended to reduce or eliminate irritability-like behavior and/or compulsive-like behavior in the patients.
  • the methods of the invention entail administering a pharmaceutical composition containing an effective amount of a nicotine degrading fusion enzyme described herein (and/or a polynucleotide sequence or expression vector encoding the enzyme) to a subject in need thereof.
  • the subject suitable for treatment is typically one afflicted with or at risk of developing one or more of the nicotine related conditions or symptoms noted above.
  • various subjects are amenable to treatment with the methods of the invention.
  • the subjects to be treated are human patients.
  • the fusion nicotine-degrading enzy me can be administered prior to intake of nicotine by the subject, during intake of nicotine by the subject, or after cessation of nicotine intake by the subject in some embodiments, the nicotine-degrading enzyme is the NicA2-Jl fusion enzyme exemplified herein or a conservatively substituted variant thereof
  • the invention provides methods for promoting nicotine cessation in a subject.
  • the methods involve administering to a subject undergoing nicotine consumption a therapeutically effective amount of a nicotine-degrading fusion enzyme described herein. Nicotine cessation is promoted or facilitated in the subject by, e.g., degrading nicotine in the body of the subject, and by reducing compulsive-like and irritability -like nicotine intake.
  • the administered nicotine-degrading fusion enzyme is NicA2-Jl exemplified herein or a variant containing one or more conservatively substituted amino acid residues.
  • the invention provides methods of treating (or reducing severity of) nicotine addiction in a subject.
  • the methods involve administering to the subject suffering from nicotine addiction a therapeutically effective amount of a nicotine-degrading fusion protein described herein.
  • Nicotine addiction is treated by, e.g., decreasing nicotine levels in the blood and brain of the subject, by reducing nicotine seeking and craving, and by ameliorating withdrawal symptoms (e.g., hyperalgesia) associated with nicotine use.
  • nicotine addiction in the subject is treated by administering an effective amount of the fusion enzyme that is sufficient to reduce compulsive-like behavior or responding.
  • Compulsive-like behavior or repetitive compulsive behavior refers to a small, restricted and repetitive behavior, which is usually not disturbing in a pathological way, and which does not necessarily lead to an actual reward or pleasure. See, e.g., Mitra et al, Front. Behav. Neurosci. 2016; 10: 244.
  • nicotine addiction in the subject is treated by administering an effective amount of the fusion enzyme that is sufficient to reduce irritability-like behavior.
  • Irritability -like behavior e.g., defensive and aggressive response
  • the administered nicotine-degrading fusion enzyme is NicA2- Jl exemplified herein or a variant containing one or more conservatively substituted ammo acid residues.
  • the invention provides methods of preventing relapse of nicotine dependence in a subject that exhibits symptoms of nicotine dependence. These methods involve administering to the subject who has previously used nicotine a
  • a nicotine-degrading fusion protein described herein Relapse of nicotine dependence is prevented by, e.g., reducing blood nicotine levels, suppressing somatic and emotional symptoms associated with nicotine withdrawal, and extending the duration of smoking abstinence.
  • relapse of nicotine dependence is prevented by administering an effective amount of the fusion enzyme that is sufficient to reduce irritability -like behavior or compulsive-like behavior in some methods, the administered nicotine-degrading fusion enzyme is NicA2-.Il exemplified herein or a variant containing one or more conservatively substituted amino acid residues.
  • the invention provides methods for reducing the toxicity of nicotine in a subject.
  • a subject in need of treatment is administered at least one fusion nicotine-degrading enzyme of the invention.
  • These methods can ameliorate the negative effects of nicotine absorption that occurs m people who smoke or chew tobacco.
  • the methods and compositions of the invention can lower the amount of nicotine that reaches or is maintained in the brain, liver, and vascular system, thereby reducing the destructive physiological effects of nicotine.
  • the administered nicotine-degrading fusion enzyme is NicA2-J! exemplified herein or a variant containing one or more conservatively substituted amino acid residues.
  • the invention further provides pharmaceutical compositions and related pharmaceutical combinations (e.g., kits) for treating nicotine addiction, preventing relapse and treating symptoms associated with nicotine withdrawal.
  • the pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation.
  • the pharmaceutical compositions or kits of the invention typically contain a therapeutically effective amount of a NicA2 fusion enzyme disclosed herein or a vector expressing the same. They can additionally include one or more pharmaceutically acceptable carrier. They may optionally also contain other therapeutic ingredients.
  • Pharmaceutically acceptable carriers can be any additives, diluents, or excipients, that are compatible with the other ingredients of the formulation, and not deleterious to the subject.
  • a therapeutically effective amount may depend on the subject being treated, the condition being treated, the desired effect, and the intended duration of the therapeutic effect.
  • the therapeutically effective amount is an amount that is effective to treat nicotine addiction, promote smoking cessation, reduce the relapse of nicotine consumption, treat nicotine poisoning m a subject in need thereof, reduce the risk of relapse of nicotine consumption, extend a duration of smoking abstinence in a subject who has quit smokmg, or increase a likelihood of long term abstinence from smoking.
  • the therapeutically effective amount is an amount that is sufficient for preventing or reducing withdrawal symptoms, e.g., hyperalgesia, irritability -like behavior and compulsive-like behavior.
  • the therapeutically effective amount is an amount that is sufficient for ameliorating or eliminating one or more symptoms associated with nicotine poisoning.
  • symptoms include, e.g., feeling queasy or throwing up, stomachache, mouthwatering, quick and heavy breathing, faster heartbeat, higher blood pressure, pale skin, headache, dizzy, off-balance, confused, diarrhea, shallow breathing, slower heartbeat, lower blood pressure, lethargy, feeling weak, slow reflexes, unable to control muscles, and seizures.
  • a therapeutically effective amount of the fusion protein or expression vector therefor may be from about 0.01 mg/kg to about 100 mg/kg, including any amount in between. Accordingly, in some embodiments, the method comprises administering from about 0.01 mg/kg to about 100 mg/kg, or any amount in between, or greater, of the nicotine- degrading fusion enzyme or expression vector therefor.
  • the method may comprise administering from about 0.1 mg/kg to about 500 to 750 mg/kg, about 0.5 mg/kg to about 300 to 500 mg/kg, about 2 mg/kg to about 100 to 300 mg/kg, about 4 mg/kg to about 50 to 100 mg/kg of body weight, or about 8 mg/'kg to about 20 to 50 mg/kg, of the nicotine- degrading fusion enzyme or expression vector therefor although other dosages may provide beneficial results.
  • the amount administered may be adjusted depending on various factors including, but not limited to, the specific enzyme, nucleic acid, vector or combination thereof being administered (including whether it is modified to enhance efficacy and/or prolong half- life); the disease or condition being treated; the weight of the subject; the physical condition of the subject (including the degree of smoking addiction, level of circulating nicotine, etc ), the health of the subject, and the age of the subject.
  • factors can be determined by employing animal models, clinical trials, or other test systems available in the art.
  • the therapeutically effective amount of the administered fusion nicotine-degrading enzyme should achieve a serum concentration of the enzyme of from about 20 nM to about 400 nM in the subject.
  • the therapeutically effective amount of the administered enzyme should achieve a serum concentration of at least 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 1 10 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, 300 nM, 310 nM, 320 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM, 390 nM, or 400 nM of the enzyme in the subject.
  • the therapeutically effective amount of the administered enzyme should achieve a serum concentration of the enzyme of from about 0.1 mM to about 100 mM, or from about 0.1 mM to about 50 mM, or from about 0.2 mM to about 50 mM, or from about 0.4 mM to about 40 mM, or from about 0.5 mM to about 10 mM in the subject.
  • the therapeutically effective amount of the enzyme or expression vector therefor administered may achieve a serum concentration of at least 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0 9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 12.0 mM, 14.0 mM, 16.0 mM, 18.0 mM, 20.0 mM, 22.0 mM, 25.0 mM, 28.0 mM, 30.0 mM, 32.0 mM, 35.0 mM, 38.0 mM
  • the dosing frequency may be selected and adj usted depending on various factors including, but not limited to, the specific enzyme, nucleic acid, vector or combination thereof being administered (including whether it is modified to enhance efficacy and/or prolong half- life); the disease or condition being treated; the weight of the subject; the physical condition of the subject (including the degree of smoking addiction, level of circulating nicotine, etc.), the health of the subject, and the age of the subject.
  • a therapeutically effective amount of the nicotine-degrading fusion enzyme is administered once daily, once every two days, once even' three days, twice weekly, thrice weekly, once weekly, once every' two weeks, once every three weeks, once every month, or once every two months, once every three months, once every' six months, or less frequently in other some embodiments, a therapeutically effective amount of the nicotine-degrading fusion enzyme is administered several times a day
  • administration of the nicotine-degrading fusion enzyme or expression vector therefor is m a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the nicotine-degrading enzymes, expression vectors, and compositions may be essentially continuous over a preselected period of time or may be in a senes of spaced doses. Both local and systemic administration is contemplated.
  • the method is effective to reduce nicotine levels in the subject in some embodiments, the method is effective to reduce serum levels of nicotine in the subject.
  • the method is effective to reduce serum levels of nicotine in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • the method is additionally or alternatively effective to reduce brain levels of nicotine in the subject.
  • the method is additionally or alternatively effective to reduce brain levels of nicotine in the subject by at least 10%, 15%, 2014, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • a higher dose is needed to achieve greater than 95% reduction of brain levels of nicotine as compared to that effective to achieve greater than 95% reduction of serum levels of nicotine, such as 2x, 4x, 8x, lOx, 20x, 3 Ox, 40x, 5 Ox, or lOOx of a dose effective to achieve greater than 95% reduction of serum levels.
  • the nicotine-degrading fusion enzyme or expression vector therefor may he administered by any a route of administration.
  • the nicotine-degrading fusion enzyme is administered by a route of administration selected from the group consisting of intranasally, orally, subcutaneously, intravenously, intrapentoneally, and intramuscularly.
  • the nicotine-degrading fusion enzyme and/or expression vector is formulation in a pharmaceutical composition suitable for the intended route of administration, as discussed in more detail below.
  • the fusion nicotine-degrading enzyme disclosed herein can be formulated as a controlled-release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel.
  • the various pharmaceutical compositions can be prepared in accordance with standard procedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19 lh Ed., Mack Publishing Company,
  • the half-life of the NicA2 variant was extended to over 5 days.
  • Nicotine dependence is characterized by the emergence of nicotine abstinence syndrome after the cessation of chronic nicotine exposure.
  • Such an abstinence syndrome has been characterized in both humans and rats and is associated with both somatic and motivational components.
  • the somatic signs of nicotine withdrawal include abdominal constrictions, facial fasciculation, ptosis, and hyperalgesia.
  • the motivational components include hyperalgesia and irritability -like behavior.
  • the hindpaw withdrawal threshold was determined by using von Frey filaments, ranging from 3.63 to 125.89 g. Testing began after 10 min of habituation to the testing environment. The series of von Frey hairs was applied from below the wire mesh to the central region of the plantar surface of the left hindpaw in ascending order, beginning with the lowest filament (3.63 g). The filament was applied until buckling of the hair occurred, and it was maintained for approximately 2 s. A withdrawal response was considered valid only if the hindpaw was completely removed from the platform. If withdrawal did not occur during three applications of a particular filament, then the next larger filament in the series was applied in a similar manner. Once the threshold was determined for the left hindpaw, the same testing procedure was repeated for the right hindpaw after 5 min. The one-way
  • Example 5 Some exemplified materials and methods
  • a 638-bp PCR fragment was amplified using primers NICA2N5 and NICA2ECOR3 (Table Sl) and pET28b-WT-NicA2 plasmid as a template.
  • the PCR fragment was gel-purified, digested by restriction endonucleases Nco I and EcoR I, and cloned back into the pET28b-WT-NicA2 vector (Nco I and EcoR I digested).
  • the A50-NicA2 protein was expressed in BL2l(DE3) E. coli cells, purified by IMAC.
  • ABDNICA5 and NICA2ECOR3 (Table 4) and plasmid pET28b-A50-NicA2 as a template.
  • the PCR fragment was gel-purified, digested by restriction endonucleases Nco I and EcoR I, and cloned back into a pET28b-A50-NicA2 vector (Nco I and EcoR I digested).
  • the NicA2- Jl protein was expressed in BL2l(DE3) E. coli cells, purified by IMAC.
  • Primer name Primer sequence (SEQ ID NO: )
  • NICA2N5 5 ATATACCATGGGTGGCTTCGATTACGATGTGGTAGTAG 3’ (1)
  • the flow cell preceding the ligand flow cell was activated by NHS/EDC and deactivated by 1.0 M ethanolamine-HCl (pH 8.5), and was served as a reference flow cell in succeeding kinetic analysis.
  • various concentrations of NicA2-Jl ranging from 31.25 to 2000.00 nM were injected randomly and individually over both reference and ligand surfaces for 5 min, then dissociated in running buffer for 30 min before the surface was regenerated with 10 mM Glycine-HCl (pH 2.2). All analyses were double referenced and conducted in duplicates. The interaction between NicA2-Jl and immobilized albumin was recorded within the sensorgram.
  • the kinetic data were evaluated via fihing the sensorgram by BIAevaluation software using a 1 : 1 (Langmuir) binding model.
  • the kinetic constants including association and dissociation rate constants (ka and kd) and equilibrium dissociation constant (KD), were summarized in Table 2 above.
  • MS operational parameters were: API-ES mode, channel 1 (90%) positive single ion monitoring (SIM) of m/z 179 (30%), 161 (30%), 166 (30%) and 163 (10%), corresponding to the M+ peak of the reaction products, labeled internal standard and substrate respectively and channel 2 (10%) scan for positive ions; nitrogen as a nebulizing and drying gas (35 psi, 12 L/min),
  • Serum samples from rats were diluted 10 folds with PBS and then coated in 96-well plate (50 pL/well) by dry method.
  • Various concentrations of pure NicA2-Jl (0, 1, 2, 5, 10, 15, 20 pg/mL, 50 pL) diluted in 10% naive rat serum/PBS was coated in the same plate for standard curve. The plate was placed at 37 ° C for overnight and fixed by methanol, then blocked with blotto (5% nonfat milk in PBS).
  • Polyclonal rabbit anti NicA2 produced by TSRI Center for Antibody Development and Production was used as the primary antibody (rabbit serum, 1: 100 dilution) and goat anti rabbit with HRP was used as secondary antibody (1 : 10000 dilution).
  • TMB Substrate Kit (ThermoFisher) was used to for signal development. The enzyme concentrations in blood were calculated based on the standard curve generated by pure NicA2-Jl.
  • the animals were housed in standard cages in a room with artificial lightning (12 h/l2 h light/dark cycle, lights off at 8:00 AM) at constant temperature (20-22 °C) and humidity (45-55%) with food and water available ad libitum.
  • the rats were handled once daily for 5 min during the first week after arrival to the vivarium. All the procedures were conducted during the dark cycle.
  • the animal procedures met the guidelines of the National Institutes of Health and were approved by The Scripps Research Institute Institutional Animal Care and Use Committee (protocol no. 08-0015). All the surgical procedures were performed under isoflurane anesthesia, and all necessary steps were taken to minimize suffering of the animals.
  • Nicotine hydrogen tartrate salt was dissolved in 0.9% sterile physiological sodium chloride and the pH was adjusted to 7.3 with NaOH 1 M.
  • the daily dose of nicotine that was delivered by the osmotic minipumps (Alzet, 2ML2, 5 pL/h) was 3.15 mg/kg.
  • IP intraperitoneally
  • Rats Males and females were divided into 4 groups (8 rats per group). Four males and four females composed every group. Two groups of rats were chronically exposed to nicotine for 7 days. Nicotine (3.15 mg/kg/day) was infused using minipumps (Alzet 2ML2) that releases 5pL of fluid/h implanted in the back underneath the skin of the rats. Two other groups of rats were chronically exposed to saline solution for 7 days that was infused using osmotic minipumps. After 12 hours of osmotic minipumps implantation, rats were daily administered (10:00 AM) with NicA2-Jl (10 mg/mL/kg) or PBS 1% as a control (IP).
  • Nicotine 3.15 mg/kg/day
  • minipumps Alzet 2ML2
  • Nicotine (3.15 mg/kg/day) was infused using osmotic minipumps (Alzet 2ML2) that release 5 pl of fluid/h implanted in the back (underneath the skin) of the rats for 7-days.
  • Endotoxin-free NicA2-Jl was dosed to rats intraperitoneally (IP) at 10 mg/mL/kg daily, with same amount of PBS as control.
  • IP intraperitoneally
  • blood was collected and immediately mixed with 4 volume of methanol (with 1 mM of nicotine D3 as internal standard) to quench the enzyme.
  • Rats were sacrificed, and brains were collected after 7 days and flash-frozen with liquid nitrogen. The brains were cut along commissure and half of each brain was weighted and used for analysis. The brain pieces were homogenized in 1 mL PBS and centrifuged at 10000 rpm for 30 min. The supernatant was mixed with same volume of 5% NH4OH, nicotine D3 added to final concentration of 0.1 mM as internal standard and then extracted with Oasis HLB 96-well pElution Plate. The elution was evaporated in Genevac and re dissolved in HEPES buffer and 2% TFA for LC-MS.
  • MS operational parameters were: API-ES mode, channel 1 (90%) positive single ion monitoring (SIM) of m/z 166 (50%, nicotine D3) and 163 (50%, nicotine) and channel 2 (10%) scan for positive ions; nitrogen as a nebulizing and drying gas (35 psi, 12 L/min), HV capillary voltage at 4 kV and the drying gas temperature to 300 ° C. To protect the detector from salts in the buffer, MS was turned on with a delay 1.4 min after injection.
  • SIM positive single ion monitoring
  • the other group was administered phosphate-buffered saline (PBS; i.p.) and run in parallel, serving as a control group.
  • PBS phosphate-buffered saline
  • Fig. 4A The rats were then given another five sessions of access to nicotine with a higher dose of NicA2-Jl (10 mg/kg), and blood was collected again at the end of the fifth session.
  • NicA2-Jl at 10 mg/kg significantly reduced blood nicotine levels (Fig. 4B).
  • the NicA2- Jl group exhibited lower defensive and aggressive responses compared with their level of irritability before treatment and compared with the PBS-treated group (Fig. 5B).
  • One possible explanation for these results is that withdrawal in NicA2-Jl -treated rats may have occurred before the withdrawal test was performed.
  • Rats that previously escalated their nicotine intake underwent an extinction phase for 21 h/day for 10 consecutive days.
  • the operant program was identical to the one that was previously used for nicotine self-administration, with the exception that responses at the drug lever did not result in nicotine delivery.
  • extinction criterion was met ( ⁇ 5 total responses in the first hour; Fig. 9A)
  • stress- and nicotine-induced reinstatement was assessed using a within-subjects design.
  • Animals with a history of PBS treatment exhibited robust reinstatement of nicotine seeking after a single intravenous injection of nicotine (0.03 mg/kg), whereas animals with a history ofNicA2-Jl treatment did not exhibit reinstatement (Fig. 9B).

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Abstract

The present invention provides nicotine-degrading fusion enzymes that are comprised of a N-terminally truncated nicotine-degrading enzyme (e.g., NicA2) and a fusion partner (e.g., an albumin binding moiety). The invention also provides therapeutic methods of using the fusion enzymes for promoting nicotine cessation, treating nicotine addiction or nicotine poisoning, and prevent relapse.

Description

COMPOSITIONS AND METHODS RELATED TO NICOTINE ADDICTION AND
CESSATION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Numbers 62/607,423 (filed December 19, 2017; now pending) and 62/765,355 (filed August 20, 2018; now pending). The full disclosures of the priority applications are incorporated herein by reference in their entirety and for all purposes.
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant numbers DA041839 and DA036691 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] Smoking is the leading cause of preventable illness in the world today with tobacco use killing 6 million people per year. Effective cessation aids are essential to assist in reducing the prevalence of cigarette smoking and related illness. In addition, nicotine withdrawal is characterized by somatic and affective symptoms, including irritability and hyperalgesia, leading to powerful craving for tobacco. Despite the existence of several approved medications, the rate of relapse in abstinent smokers remains high after 12 months (-75-80%). Pharmacokinetic approaches to prevent nicotine from reaching the brain have been tested using vaccines, but such efforts have failed because antibody titers are not sufficient to prevent nicotine from reaching the brain. An alternative strategy to treat tobacco use disorder is to reduce the psychoactive effects of nicotine by preventing nicotine from reaching the brain. Such an approach may allow a progressive reduction of the level of dependence, leading to a decrease in craving and preventing relapse that is induced by reexposure to nicotine.
[0004] There is an unmet need in the art for novel and more effective approaches to promote nicotine cessation, reduce the psychoactive effects of nicotine, decrease craving, and prevent relapse. The present invention is directed to these and other needs. SUMMARY OF THE INVENTION
[0005] The invention relates to methods and compositions for promoting nicotine cessation and for treating nicotine addiction. In one aspect, the invention provides novel nicotine-degrading fusion proteins that can degrade nicotine in vivo and treat disorders related to nicotine consumption and addiction. The nicotine-degrading fusion proteins of the invention contain aN-terminal truncated nicotine-degrading enzyme and an albumin binding moiety. In some nicotine-degrading fusion proteins of the invention, the nicotine-degrading fusion enzyme is NicA2 isolated from Pseudomonas putida S16 or a conservatively substituted variant thereof. In some nicotine-degrading fusion proteins of the invention, the albumin binding moiety is albumin binding domain (ABD)035 or a conservatively substituted variant thereof. In some embodiments, the employed N-terminally truncated nicotine degrading enzyme is a NicA2 variant with a deletion of about 25 or more N-terminal amino acid residues of the wildtype enzyme. In some of these embodiments, the deletion is about 45, 46, 47, 48, 49, 50, 51 or more N-terminal amino acid residues.
[0006] In some nicotine-degrading fusion proteins of the invention, the albumin binding moiety is fused to the N-terminus of the truncated enzyme. Some nicotine-degrading fusion proteins of the invention contain aNicA2 variant with a deletion of about the first 50 N- terminal residues, and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof. In some of these embodiments, the fusion enzyme has substantially the same or better substrate binding and/or catalytic activity relative to the wildtype NicA2 enzyme. In some nicotine-degrading fusion proteins of the invention, the truncated enzyme is linked to the albumin binding moiety via a linker moiety.
[0007] In a related aspect, the invention provides polynucleotide sequences encoding a nicotine-degrading fusion protein that contains a N-terminal truncated nicotine-degrading fusion enzyme and an albumin binding moiety. In some embodiments, the encoded nicotine degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
[0008] In another aspect, the invention provides methods of treating nicotine addiction and/or promoting nicotine cessation in a human patient suffering from nicotine addiction and/or nicotine consumption. These methods entail administering to the patient in need of treatment a therapeutically effective amount of a nicotine-degrading fusion protein described herein. In some embodiments, the employed nicotine-degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof. In some of the methods, the effective amount of the administered enzyme is an amount that is sufficient to reduce compulsive-like behavior and/or irritability -like behavior in the patient.
[0009] In another aspect, the invention provides methods for preventing relapse of nicotine dependence in a human patient exhibiting symptoms of nicotine dependence. These methods involve administering to the patient a therapeutically effective amount of a nicotine degrading fusion protein described herein. In some embodiments, the administered nicotine degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof. In some of these methods, the effective amount of the administered enzyme is an amount that is sufficient to reduce compulsive-like behavior and/or irritability -like behavior in the patient.
[0010] In another related aspect, the invention provides methods for treating nicotine poisoning in a human patient in need thereof. The methods involve administering to the patient a therapeutically effective amount of a nicotine-degrading fusion protein described herein. In some methods, the administered nicotine-degrading fusion protein contains nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof. In some embodiments, the effective amount of the administered enzyme is an amount that is sufficient to ameliorate one or more symptoms associated with nicotine poisoning.
[0011] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.
DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 shows pharmacokinetics of NicA2 and NicA2-Jl in rat serum.
[0013] Figure shows effect of NicA2-Jl during nicotine withdrawal in rats. NicA2-Jl blocks total withdrawal score (A), hyperalgesia (B) irritability like behavior (C) and during withdrawal. (Sal: saline; Nico: nicotine).
[0014] Figure 3 shows nicotine concentrations in rat blood (A) after 1 or 5 days and brains (B) after 7 days of NicA2-Jl administration. (ND: not detected). [0015] Figure 4 shows blood nicotine levels after escalation of nicotine intake. Detailed timeline of the experiments (upper panel). (A) After the last session of escalation of nicotine intake, blood nicotine levels were detected in rats that were pretreated with NicA2-Jl (2 mg/kg) and rats that were pretreated with PBS. Student’s /-test showed no difference in blood nicotine levels between groups (/ = 1.15, df = 14,/.? > 0.05). (B) At the termination of escalation of nicotine intake, rats that were pretreated with NicA2-Jl (10 mg/kg) exhibited nearly undetectable levels of nicotine in blood compared with the PBS -pretreated group (Student’s /-test; / = 4.198, df = 14, p < 0.001). **p < 0.01.
[0016] Figure 5 shows that NicA2-Jl prevents nicotine-addiction like behavior during withdrawal. Detailed timeline of the experiments (upper panel). (A) NicA2-Jl suppressed hyperalgesia during nicotine withdrawal. The two-way mixed-factorial ANOVA, with group (PBS vs. NicA2-Jl) as the between-subjects factor and time (baseline, pre-escalation hyperalgesia, and post-escalation hyperalgesia) as the within-subjects factor, revealed significant effects of group (Fi,i4 = 4.91, p = 0.04) and time ( 2,i4 =10.89 ,p = 0.0003) and a significant group x time interaction (/’2.2s = 5.11, » = 0.012). The Newman-Keuls post hoc analysis revealed that mechanical sensitivity significantly decreased during withdrawal (pre escalation) in PBS-treated rats compared with their mechanical sensitivity before nicotine exposure (p = 0.017) and compared with the NicA2-Jl group (p = 0.007). Mechanical sensitivity after pretreatment withNicA2-Jl was comparable to baseline sensitivity before nicotine exposure (p > 0.05), suggesting thatNicA2-Jl treatment completely reversed withdrawal-induced hyperalgesia (pre-escalation vs. post-escalation,/? = 0.043), whereas this effect was not detected in the PBS group, which exhibited more severe hyperalgesia during withdrawal (post-escalation vs. BSL, p = 0.0018). Moreover, the Newman-Keuls post hoc analysis showed that hyperalgesia during post-escalation was completely reversed in the NicA2-Jl group (post-escalation in the PBS group vs. post-escalation in the NicA2-Jl group, p = 0.041). (B) Effect of NicA2-Jl on irritability-like behavior, reflected by defensive and aggressive responses. The baseline of defensive and aggressive responses was measured during 48 h of spontaneous nicotine withdrawal before treatment with NicA2-Jl or PBS. All of the other measures were performed during 48 h of spontaneous nicotine withdrawal after the last escalation phase after the completion of NicA2-Jl treatment (10 mg/kg). A significant decrease in defensive responses (n = 8; / = 4.5, df = 7 ,p < 0.01) and aggressive responses ( n = 8; / = 5.22, df = 7, > < 0.01) was observed in NicA2-Jl -pretreated rats. No changes from baseline were observed in the PBS group. Student’s paired /-test revealed a significant reduction of aggressive responses 0 = 2.27, df = 14, p < 0.05) but not defensive responses 0 = 1.85, df = 14 , p > 0.05) in NicA2-Jl -pretreated rats compared with PBS-pretreated rats. *p < 0.05, **p < 0.01, vs. baseline; #p < 0.05, vs. pre-escalation; &/> < 0.05, post-escalation comparisons between PBS and NicA2-Jl groups.
[0017] Figure 6 shows that acute administration of NicA2-Jl decreased withdrawal- induced hyperalgesia. Detailed timeline of the experiments (upper panel). (A) Mechanical nociceptive thresholds immediately after nicotine escalation and 48 h into withdrawal. During withdrawal, a significant decrease in hyperalgesia thresholds was observed compared with baseline thresholds (n = 11; t = 11.9, df = 10, p < 0.01). (B) In PBS-treated animals, a significant decrease in hyperalgesia thresholds was observed compared with baseline thresholds at 24 h into withdrawal (n = 11; t = 13.89, df = 10, p < 0.01) and 48 h into withdrawal (n = 11; / = 9.96, df = 10, p < 0.01) but not during nicotine self-administration.
(C) In NicA2-Jl -treated rats, a decrease in hyperalgesia thresholds was observed at 24 h into withdrawal (n = 11; t = 13.89, df = 10, p < 0.05). *p < 0.05, **p < 0.01.
[0018] Figure 7 shows effect of NicA2-Jl on the escalation of nicotine intake (1 and 21 h). (Top) Detailed timeline of the experiments (upper panel). (A) Nicotine self-administration and escalation of nicotine intake during the first hour of nicotine exposure. Two separate one way ANOVAs showed that the animals significantly escalated their nicotine intake in the first hour of the session during escalation in the PBS group ( is.ios = 9.92 ,p = 0.0001) and NicA2-Jl group ( is.ios = 51.691 , p = 0.0001). The Newman-Keuls post hoc analysis revealed significant escalation on the last three intermittent-access days compared with the last three continuous-access days (p = 0.001) in both groups. (B) Inactive lever responses did not change over time in either the PBS group ( is.ios = 0.62 ,p = 0.54) or NicA2-Jl group ( i5,io5 = 019, p = 0.32). (C) The two-way mixed-factorial ANOVA, with treatment (PBS and NicA2-Jl) as the between-subjects factor and time as the within-subjects factor, did not show a significant treatment x time interaction (/’io.uo = 0.991 ,p = 0.45). (D) Inactive lever responding was unaffected by NicA2-Jl treatment (/’io.uo = 0.782, = 0.55). (E) Nicotine self-administration and escalation of nicotine intake during the 21 h of nicotine exposure.
Two separate one-way ANOVAs showed that the animals significantly escalated their nicotine intake in the PBS group ( is.ios = 5.53, p = 0.0001) and NicA2-Jl group (/’U. HU = 4.186,/ = 0.001). The Newman-Keuls post hoc analysis indicated significant escalation on the last three intermittent-access days compared with the last three continuous-access days (p = 0.01) in both groups. (F) Inactive lever responses did not change over time in either the PBS group ( i5,io5 = 0.32 , p = 0.23) or NicA2-Jl group (T¾,ΐ05 = 0.49, p = 0.41). (G) The two-way mixed-factorial ANOVA, with treatment (PBS and NicA2-Jl) as the between- subjects factor and time as the within-subjects factor, showed a significant effect of time (Fio,i4 = 2.37 ,p = 0.013) but no treatment x time interaction ( IO.MO = 0.991, p = 0.45). (H) Inactive lever responding was unaffected by NicA2-Jl treatment (/'in.un = 0.65 ,p = 0.76)
[0019] Figure 8 shows that NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats. Detailed timeline of the experiments (upper panel). (A) When footshock (0.1 mA) was introduced, the results showed that nicotine-dependent animals that exhibited escalation of nicotine intake and were pretreated with PBS continued responding for nicotine despite the adverse consequences of footshocks at a higher level compared with their intake prior to footshock exposure (« = 8; / = 3.063, df = 7 , p = 0.028). No change in responding was observed in NicA2-Jl (10 mg/kg)-pretreated rats (n = 8: / = 0.39, df = 7 , p = 0.7).
Student’s /-test revealed a significant difference between groups (/ = 2.315, df = 14, p =
0.04). (B) When a higher footshock intensity (0.2 mA) was introduced, the results showed that nicotine-dependent animals that were pretreated with PBS exhibited no changes in responding for nicotine despite the adverse consequences of footshocks compared with their intake prior to footshock exposure (/ = 1.88, df = Ί, r = 0.118), whereas a significant reduction was observed in NicA2-Jl -pretreated rats (/ = 3.78, df = 7 , p = 0.0091). Student’s /- test revealed a significant difference between groups (/ = 3.5, df = 14, p = 0.0048). *p < 0.05, **p < 0.01, vs. baseline; #p < 0.05, up < 0.01, comparison between PBS and NicA2-Jl groups.
[0020] Figure 9 shows that NicA2-Jl prevents nicotine- and stress-induced reinstatement after extinguished nicotine intake. Detailed timeline of the experiments (upper panel). (A) Extinction phase. (B) Pretreatment with NicA2-Jl (10 mg/kg) prevented nicotine prime- induced reinstatement of nicotine-seeking behavior. The two-way mixed-factorial ANOVA, with group (PBS v.v. NicA2-Jl) as the between-subjects factor and treatment (extinction, vehicle, and 0.03 mg/kg/injection nicotine) as the within-subjects factor, showed a significant effect of treatment (/' 2. i 4 = 9.14 ,p = 0.0008) and a significant group x treatment interaction (7 2,28 = 3.48, p = 0.044). The Newman-Keuls post hoc analysis revealed that one injection of nicotine (0.03 mg/kg/injection) significantly increased the number of lever presses compared with the extinction phase in PBS-treated rats (p < 0.0008). Nicotine priming did not induce the reinstatement of nicotine-seeking behavior in rats that were pretreated with NicA2-Jl during escalation (nicotine priming vs. extinction, = 0.61). The post hoc analysis showed that nicotine-induced priming was abolished in the NicA2-Jl group compared with the PBS group (nicotine priming in the PBS group vs. nicotine priming in the NicA2-Jl group ,p = 0.047). (C) Inactive lever responses were unaffected by nicotine infusions in either group (/’2.2s =
0.64 , p = 0.53). (D) NicA2-Jl (10 mg/kg) prevented the stress (yohimbine)-induced reinstatement of nicotine-seeking behavior in the PBS -pretreated group. The two-way mixed- factorial ANOVA, with group (PBS vs. NicA2-Jl) as the between-subjects factor and treatment (extinction, vehicle, and yohimbine) as the within-subjects factor, showed a significant effect of treatment (/' 2.14 = 3.3 1. p = 0.05) and a significant group x treatment interaction (F2, 28 = 5.12, p = 0.0127). The Newman-Keuls post hoc analysis revealed that yohimbine significantly increased the number of lever presses in the PBS-pretreated group compared with the extinction phase (p = 0.011). No effect of yohimbine-induced reinstatement was observed in the NicA2-Jl pretreated group. (E) Inactive lever responses were unaffected by yohimbine in either group Fi, 28 = 0.15 , p = 0.85). *p < 0.05, **p < 0.01, vs. extinction; &p < 0.05, nicotine priming-induced reinstatement between PBS and NicA2-Jl groups.
DETAILED DESCRIPTION
[0021] The present invention is predicated in part on the studies undertaken by the inventors to develop nicotine-degrading enzymes suitable for clinical uses in human subjects. In contrast to simple antibody-mediated sequestering of nicotine entering the brain, the inventors explored an alternative strategy to reducing nicotine’s brain concentration. By decreasing nicotine’s peripheral circulation through nicotine catabolism mediated by nicotine-degrading enzymes, this alternative strategy ensures that an effective concentration could not be reached or maintained in the brain. As detailed herein, the nicotine-degrading enzymes developed by the inventors can reverse nicotine dependence, decrease compulsive- like intake, and prevent relapse in a translational animal model of nicotine addiction.
Moreover, the enzymes (e.g., the NicA2-Jl fusion protein exemplified herein) have a favorable pharmacokinetic profile, including a relatively long half-life and simple route of administration.
[0022] As exemplification, the inventors developed a fusion protein based on a truncated nicotine degrading enzyme NicA2 and albumin binding domain (ABD)035, which demonstrated significantly improved in vivo stability. The exemplified fusion protein, NicA2-Jl, has demonstrated surprisingly advantageous properties relative to NicA2 or variants that are known in the art. NicA2-Jl was found to have catalytic activity that is similar to the wildtype enzyme NicA2 but substantially improved serum stability. The fusion enzyme is able to degrade nicotine and prevents the development of nicotine dependence in rats. Serum nicotine distribution and behavioral testing revealed that the fusion enzyme completely eliminates blood nicotine content, thereby halting the drug’s psychoactive effects. The inventors also conducted a series of studies to determine whether NicA2-Jl has translational relevance and prevents addiction-like behaviors in animals with a history of compulsive-like nicotine self-administration. It was demonstrated that NicA2-Jl decreased blood nicotine levels and had remarkable preclinical efficacy in reducing addiction-like behaviors in nicotine-dependent rats. NicA2-Jl administration reduced blood nicotine levels, reversed somatic and emotional signs of nicotine withdrawal in dependent rats, reduced compulsive-like responding for nicotine, and prevented nicotine- and stress-induced relapse.
[0023] As demonstrated herein, NicA2-Jl dose-dependently affected nicotine clearance in blood. At a low dose (2 mg/kg), blood nicotine levels did not significantly decrease, which may reflect a compensatory effect of repeated nicotine intake. However, at a higher dose (10 mg/kg), NicA2-Jl degraded nicotine in blood to undetectable levels in all of the rats (< 5 nM), with the exception of one rat that still had 25% of blood nicotine relative to controls. Such a robust decrease in blood nicotine levels is remarkable and vastly superior to previous approaches that used immunopharmacotherapies. The results exemplified herein
demonstrated a > 95% decrease in nicotine in both blood and brain at the higher dose, and indicate that the decrease in nicotine levels was associated with a reversal of key behavioral symptoms of nicotine dependence. Nicotine-dependent rats that were treated with NicA2-Jl exhibited a lack of hyperalgesia and a robust decrease in irritability -like behavior after 2 weeks of treatment. Moreover, acute NicA2-Jl treatment did not precipitate withdrawal during nicotine self-administration and decreased withdrawal-induced hyperalgesia when administered acutely. These results suggest that although NicA2-Jl degraded most of the nicotine in blood, a slight amount of nicotine likely remained in blood that was sufficient to prevent withdrawal in rats while decreasing the level of dependence.
[0024] Further, NicA2-Jl prevented the development of nicotine dependence and reversed nicotine dependence by normalizing somatic and emotional signs of nicotine withdrawal in only 2 weeks. This is a critical result because irritability during abstinence is often mentioned by users as one of the primary reasons why they relapse. Even more compelling from a translational perspective, NicA2-JI decreased compulsive-like responding for nicotine, reflected by nicotine intake despite the adverse consequences of contingent footshocks. This suggests that treatment with the NicA2-Jl enzyme reduced symptoms of nicotine withdrawal and diminished the incentive value of nicotine, thereby decreasing the motivation to take nicotine when confronted with adverse consequences. NicA2-Jl did not affect nicotine self-administration when no adverse consequences were presented, suggesting that the very low blood nicotine levels were sufficient to serve as a discriminative stimulus but not sufficient to have incentive value, including responding despite adverse
consequences.
[0025] Moreover, NicA2-Jl reduced compulsive-like nicotine seeking, which is highly relevant to the human condition, in which smoking is often associated with significant adverse social consequences (e.g., conflicts with partners) and health consequences (e.g., coughing, pulmonary disease, and cancer) that are often ignored by smokers because of the high incentive value of nicotine and the ability of nicotine to provide relief from withdrawal symptoms. The studies described herein indicate that chronic NicA2-Jl treatment can help reduce withdrawal symptoms and reduce compulsive nicotine seeking and taking.
[0026] In accordance with these exemplified studies, the invention provides nicotine degrading fusion enzymes that are comprised of aN-terminally truncated nicotine degrading enzyme and a fusion partner that enhances the stability and in vivo half-life of the enzyme while maintaining its enzymatic activities. The invention also provides polynucleotide sequences that encode the fusion enzymes described herein, as well as vectors harboring such that harbor the polynucleotide sequences for expressing the fusion enzymes. The invention further provides methods of using the fusion enzymes, the encoding polynucleotides and expression vectors in various therapeutic and prophylactic applications for countering nicotine use related medical conditions and symptoms. The various methods described herein are directed to treating nicotine addiction, treating nicotine-addiction related disorders, reducing the risk of relapse of nicotine consumption, promoting smoking cessation, extending a duration of smoking abstinence in a subject who has quit smoking, increasing a likelihood of long-term abstinence from smoking, and/or rescuing a subject from relapse of nicotine consumption.
[0027] Unless otherwise specified herein, the nicotine degrading fusion enzymes of the invention, the encoding polynucleotides, expression vectors and host cells, as well as the related therapeutic applications, can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; lst edition (1997) (ISBN-13: 978- 0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rd ed., 2000); Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al, ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Bames editors, Academic Press, lst edition, 1998). The following descriptions provide additional guidance for practicing the
compositions and methods of the present invention.
[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and
Technology, Morris (Ed.), Academic Press (Ist ed., 1992); Oxford Dictionary of
Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (Ist ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994);
Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4th ed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.
[0029] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example,“a nicotine degrading fusion enzyme" can refer to both single or plural nicotine degrading fusion enzymes, and can be considered equivalent to the phrase "at least one nicotine degrading fusion enzyme."
[0030] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
[0031] For polypeptide sequences,“conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0032] As used herein, a fusion protein is a protein molecule containing amino acid sequence from at least two unrelated proteins that have been joined together, e.g., via a peptide bond, to make a single protein. The unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence. As used herein, proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell).
[0033] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443,
1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp,
Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al, Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al, Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
[0034] The term "subject" refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms“patient” or“subject” are used herein interchangeably. Preferably, the subject is human.
[0035] The term“treating” or“alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease or condition (e.g., nicotine addiction or withdrawal symptom), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder.
Treatment may be prophylactic (to prevent or delay the onset of the disease or condition, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.
[0036] The invention provides nicotine degrading fusion enzymes and clinical applications of the same. The nicotine degrading fusion enzymes of the invention are comprised of aN-terminally truncated nicotine-degrading enzyme and a fusion partner for enhanced stability and in vivo half-life. In some embodiments, the employed nicotine degrading enzyme for truncation is obtained from bacterium Pseudomonas puiida. In some of these embodiments, the nicotine-degrading enzyme for truncation is NicA2. NicA2 was originally isolated from Pseudomonas putida (Strain 16). See, e.g., Tang et al, PLoS Genet. 9:e1003923, 2013; Tang et al., J. Bacteriol. 193:5541-42, 201 1 ; Yu et al., Sci. Rep. 4:5397, 2014; and Xue et al, J. Am. Chem Soc. 137: 10136-9, 2015. Polynucleotide sequence encoding the wildtype NicA2 enzyme is available as NCBI accession number CP002870.1 and also described in the art, e.g., W02017023904. In some specific embodiments, the employed nicotine-degrading enzyme for truncation is NicA2 as described in
WO2017023904.
[0037] The variant NicA2 enzyme used in the fusion protein of the invention typically has a N-terminal truncation relative to the wildtype enzyme. In some embodiments, the truncation comprises a deletion of about the first 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more of the N-terminal amino acid residues. In some of these embodiments, the truncation constitutes a deletion of the first 50 N-terminal residues of the wildtype NicA2 (i.e., D50- NicA2). In some other embodiments, the truncation constitutes a deletion of the first 41, 42, 43, 44, 45, 46, 47, 48, or 49 N-terminal residues of the wildtype NicA2. In still some other embodiments, the truncation constitutes a deletion of the first 51, 52, 53, 54 or 55 N-terminal residues of the wildtype NicA2 enzyme. In addition to the N-terminal truncation, the employed variant enzyme can have one or more of the amino acid residues that are different from what is present in the wildtype nicotine-degrading enzyme. In some embodiments, a variant nicotine-degrading enzyme has at least 75%, 80%, 85%, 90% or 95% sequence identity with the amino acid sequence of the wildtype nicotine-degrading enzyme, such as NicA2. In some embodiments, the NicA2 variant enzyme has at least 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 1. in some embodiments, the NicA2 variant enzyme has at least 96%, 97%, 98% or at least 99% sequence identity with the wildtype NicA2 enzyme.
[0038] In some embodiments, the employed NicA2 variant enzyme contains one or more conservatively substituted amino acid residues relative to the wildtype enzyme. Other than the structural variations noted above, the variant nicotine-degrading enzyme used in constructing the fusion protein of the invention, e.g., a conservatively substituted variant of A50-NicA2, should maintain substantially the same enzymatic function or in vivo nicotine degrading activity of the wildtype protein. For example, the employed variant should have substantially the same or better substrate binding and catalytic activity relative to the wildtype NicA2 enzyme or the \50-NicA2 variant exemplified herein. The enzy matic function of the vanant enzymes, e.g., kcat and Km values, can be readily determined via any of the in vitro or in vivo assay s exemplified herein or that is well known in the art. See, e.g., WO2017023904.
[0039] The fusion enzyme of the invention contains the N-terminal truncated nicotine degrading enzyme (e.g., \50-NicA2 exemplified herein) that is linked to a fusion partner.
The fusion partner can be any moiety that increases the circulating half-life of the truncated nicotine-degrading enzyme in vivo, e.g., an albumin-binding moiety, an albumin moiety or a polyethylene glycol moiety. In some embodiments, the employed fusion partner is an albumin-binding moiety. As exemplified herein, some fusion enzymes of the invention contain the N-terminal truncated NicA2 that is fused to albumin-binding domain ABD(035). ABD(035) is an albumin binding protein domain that is well known in the art. See, e.g., Jonsson et al., Protein Eng. Des. Sel. 2l(8):515-27, 2008. The fusion partner can be conjugated or linked to the truncated enzyme at either the N-terminus or the C-terminus of the enzyme. In some embodiments, the fusion partner, e.g., ABD(035), is fused to the N- terminus of the enzyme.
[0040] The method for generating the fusion protein of the invention is not subject to any particular limitation. The fusion protein of the invention may be a fusion protein synthesized by chemical synthesis, or a recombinant fusion protein produced by a genetic engineering technique. If the fusion protein of the invention is to be chemically synthesized, synthesis may be carried out by, for example, the Fmoc (fluorenylmethyloxy carbonyl) process or the tBoc (t-butyloxy carbonyl) process. In addition, peptide synthesizers available from, for example, Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Syntheceh-Vega, PerSeptive and Shimadzu Corporation may be used for chemical synthesis. In some preferred embodiments, the fusion proteins of the invention are produced by genetic engineering using the conventional recombination techniques routinely practiced in the art. Such techniques are described, e.g., in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rd ed., 2000); and Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
[0041] Recombinant production of the fusion enzymes of the invention typically involves removing the stop codon from a polynucleotide sequence (e.g., a cDNA sequence) coding for the fusion partner (e.g., an albumin binding protein), then appending a polynucleotide sequence (e.g., a cDNA sequence) encoding the truncated nicotine-degrading enzyme (e.g., \50-NicA2) in frame through ligation or overlap extension PCR. The fusion protein can then be expressed by inserting the resulting polynucleotide sequence encoding the fusion protein into a suitable expression system. To ensure proper folding and maintain the biological activities of the fusion partners, a linker moiety or spacer peptide may be used for linking the two components of the fusion enzymes. Such linker moieties (e.g., GS linker or G4S linker) for generating fusion proteins are well known and routinely used in the art. See, e.g., Chen et al, Adv. Drug Deliv. Rev. 2013; 65: 1357-1369; Chen et al, J. Mol. Microbiol. Biotechnol. 2017; 27: 64-71; and Guo et al, Mol. BioSyst, 2017, 13: 598. The fusion enzyme proteins of the invention may additionally include a peptide sequence for purification. Peptide sequences for purification that may be used are also known in the art. Examples of peptide sequences for purification include histidine tag sequences having an amino acid sequence in which at least four, and preferably at least six, continuous histidine residues, and the amino acid sequence of the glutathione-binding domain in glutathione S- transferase.
[0042] Thus, in some embodiments, the N-terminally truncated NicA2 fusion enzymes of the invention are generated by recombinantly linking an albumin binding protein domain to the N-terminus of the truncated NicA2 enzyme. Some specific exemplifications are discussed in detail in the Examples below. For example, construction of the fusion enzyme containing the N-truncated nicotine-degrading enzyme (e.g., \50-NicA2) to a fusion protein partner (e.g., ABD(035)) can be readily carried via the methods exemplified herein or standard protocols of biochemistry and molecular biology. Cloning and recombinant expression of truncated NicA2 and the albumin binding protein domain can be performed with techniques exemplified herein. Fusion of the enzyme to the albumin binding domain can be similarly carried out as exemplified herein for the generation of the NicA2-Jl fusion enzyme which contains the ABD(035) domain fused to the N-terminus of \50-NicA2.
[0043] In addition to the nicotine-degrading fusion proteins described above, related embodiments of the invention include polynucleotide sequences that encode such fusions, expression constructs for expressing the fusion proteins, and host cells that harbor the polynucleotides or expression constructs. The polynucleotide sequences of the invention can be any polynucleotide having a nucleotide sequence that encodes the fusion protein of the invention, although DNA is preferred. The recombinant constructs or expression vectors of the invention harbor a polynucleotide sequence of the invention that encodes a N-terminally truncated nicotine-degrading fusion enzyme. The recombinant constructs of the invention may be obtained by ligating (inserting) the polynucleotide (DNA) of the invention into a suitable vector. More specifically, the recombinant vector may be obtained by cleaving purified polynucleotide (DNA) with a suitable restriction enzyme, then inserting the cleaved polynucleotide to a restriction enzyme site or multicloning site on a suitable vector, and ligating the polynucleotide to the vector. The vector for inserting the polynucleotide sequence is not subject to any particular limitation, provided it is capable of replication in an appropriate host. The expression vectors can be, for example, bacteriophages, plasmids, cosmids or phagemids. Examples of recombinant bacteriophage or phagemid vectors include that based on a filamentous phage such as Ml 3. Plasmid vectors include those based on plasmids from, e.g., E. coli (e.g., pBR322, pBR325, pUCH8 and pUCH9), plasmids from Bacillus subtilis (e.g., pUBl lO and pTP5), and plasmids from yeasts (e.g., YEpl3, YEp24 and YCp50). The expression vectors can also include vectors derived from animal viruses such as retroviruses, vaccinia viruses and insect viruses (e.g., baculoviruses).
[0044] The invention also provides therapeutic and prophylactic methods for treating nicotine toxicity, nicotine addiction, promoting smoking cessation, reducing the relapse of nicotine consumption, preventing or reducing symptoms associated with nicotine withdrawal, or treating nicotine poisoning. In some embodiments, treatment via methods of the invention is intended to reduce or eliminate irritability-like behavior and/or compulsive-like behavior in the patients. In general, the methods of the invention entail administering a pharmaceutical composition containing an effective amount of a nicotine degrading fusion enzyme described herein (and/or a polynucleotide sequence or expression vector encoding the enzyme) to a subject in need thereof. The subject suitable for treatment is typically one afflicted with or at risk of developing one or more of the nicotine related conditions or symptoms noted above. Thus, various subjects are amenable to treatment with the methods of the invention.
Preferably, the subjects to be treated are human patients. In the practice of the various therapeutic or prophylactic methods of the invention, the fusion nicotine-degrading enzy me can be administered prior to intake of nicotine by the subject, during intake of nicotine by the subject, or after cessation of nicotine intake by the subject in some embodiments, the nicotine-degrading enzyme is the NicA2-Jl fusion enzyme exemplified herein or a conservatively substituted variant thereof
[0045] in some embodiments, the invention provides methods for promoting nicotine cessation in a subject. The methods involve administering to a subject undergoing nicotine consumption a therapeutically effective amount of a nicotine-degrading fusion enzyme described herein. Nicotine cessation is promoted or facilitated in the subject by, e.g., degrading nicotine in the body of the subject, and by reducing compulsive-like and irritability -like nicotine intake. In some methods, the administered nicotine-degrading fusion enzyme is NicA2-Jl exemplified herein or a variant containing one or more conservatively substituted amino acid residues.
[0046] In some embodiments, the invention provides methods of treating (or reducing severity of) nicotine addiction in a subject. The methods involve administering to the subject suffering from nicotine addiction a therapeutically effective amount of a nicotine-degrading fusion protein described herein. Nicotine addiction is treated by, e.g., decreasing nicotine levels in the blood and brain of the subject, by reducing nicotine seeking and craving, and by ameliorating withdrawal symptoms (e.g., hyperalgesia) associated with nicotine use. In some of the methods, nicotine addiction in the subject is treated by administering an effective amount of the fusion enzyme that is sufficient to reduce compulsive-like behavior or responding. Compulsive-like behavior or repetitive compulsive behavior (e.g., compulsive nicotine seeking and taking) refers to a small, restricted and repetitive behavior, which is usually not disturbing in a pathological way, and which does not necessarily lead to an actual reward or pleasure. See, e.g., Mitra et al, Front. Behav. Neurosci. 2016; 10: 244. In some of the methods, nicotine addiction in the subject is treated by administering an effective amount of the fusion enzyme that is sufficient to reduce irritability-like behavior. Irritability -like behavior (e.g., defensive and aggressive response) is a technical term that is also well known and characterized in the art. See, e.g., Kimbrough et al, Alcohol Clin. Exp. Res. 41, 1886- 1895, 2017. in some methods the administered nicotine-degrading fusion enzyme is NicA2- Jl exemplified herein or a variant containing one or more conservatively substituted ammo acid residues.
[0047] In some embodiments, the invention provides methods of preventing relapse of nicotine dependence in a subject that exhibits symptoms of nicotine dependence. These methods involve administering to the subject who has previously used nicotine a
therapeutically effective amount of a nicotine-degrading fusion protein described herein. Relapse of nicotine dependence is prevented by, e.g., reducing blood nicotine levels, suppressing somatic and emotional symptoms associated with nicotine withdrawal, and extending the duration of smoking abstinence. In some of these methods, relapse of nicotine dependence is prevented by administering an effective amount of the fusion enzyme that is sufficient to reduce irritability -like behavior or compulsive-like behavior in some methods, the administered nicotine-degrading fusion enzyme is NicA2-.Il exemplified herein or a variant containing one or more conservatively substituted amino acid residues.
[0048] in some embodiments, the invention provides methods for reducing the toxicity of nicotine in a subject. In these methods, a subject in need of treatment is administered at least one fusion nicotine-degrading enzyme of the invention. These methods can ameliorate the negative effects of nicotine absorption that occurs m people who smoke or chew tobacco. By decreasing the amount of nicotine in circulation throughout the body, the toxicity and psychoactive effects of nicotine are reduced. Hence, the methods and compositions of the invention can lower the amount of nicotine that reaches or is maintained in the brain, liver, and vascular system, thereby reducing the destructive physiological effects of nicotine. In some methods, the administered nicotine-degrading fusion enzyme is NicA2-J! exemplified herein or a variant containing one or more conservatively substituted amino acid residues. [0049] The invention further provides pharmaceutical compositions and related pharmaceutical combinations (e.g., kits) for treating nicotine addiction, preventing relapse and treating symptoms associated with nicotine withdrawal. The pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation. The pharmaceutical compositions or kits of the invention typically contain a therapeutically effective amount of a NicA2 fusion enzyme disclosed herein or a vector expressing the same. They can additionally include one or more pharmaceutically acceptable carrier. They may optionally also contain other therapeutic ingredients. Pharmaceutically acceptable carriers can be any additives, diluents, or excipients, that are compatible with the other ingredients of the formulation, and not deleterious to the subject.
[0050] A therapeutically effective amount may depend on the subject being treated, the condition being treated, the desired effect, and the intended duration of the therapeutic effect. In various embodiments, the therapeutically effective amount is an amount that is effective to treat nicotine addiction, promote smoking cessation, reduce the relapse of nicotine consumption, treat nicotine poisoning m a subject in need thereof, reduce the risk of relapse of nicotine consumption, extend a duration of smoking abstinence in a subject who has quit smokmg, or increase a likelihood of long term abstinence from smoking. In some embodiments, the therapeutically effective amount is an amount that is sufficient for preventing or reducing withdrawal symptoms, e.g., hyperalgesia, irritability -like behavior and compulsive-like behavior. In some embodiments, the therapeutically effective amount is an amount that is sufficient for ameliorating or eliminating one or more symptoms associated with nicotine poisoning. Such symptoms include, e.g., feeling queasy or throwing up, stomachache, mouthwatering, quick and heavy breathing, faster heartbeat, higher blood pressure, pale skin, headache, dizzy, off-balance, confused, diarrhea, shallow breathing, slower heartbeat, lower blood pressure, lethargy, feeling weak, slow reflexes, unable to control muscles, and seizures.
[0051] In general, a therapeutically effective amount of the fusion protein or expression vector therefor may be from about 0.01 mg/kg to about 100 mg/kg, including any amount in between. Accordingly, in some embodiments, the method comprises administering from about 0.01 mg/kg to about 100 mg/kg, or any amount in between, or greater, of the nicotine- degrading fusion enzyme or expression vector therefor. For example, the method may comprise administering from about 0.1 mg/kg to about 500 to 750 mg/kg, about 0.5 mg/kg to about 300 to 500 mg/kg, about 2 mg/kg to about 100 to 300 mg/kg, about 4 mg/kg to about 50 to 100 mg/kg of body weight, or about 8 mg/'kg to about 20 to 50 mg/kg, of the nicotine- degrading fusion enzyme or expression vector therefor although other dosages may provide beneficial results. The amount administered may be adjusted depending on various factors including, but not limited to, the specific enzyme, nucleic acid, vector or combination thereof being administered (including whether it is modified to enhance efficacy and/or prolong half- life); the disease or condition being treated; the weight of the subject; the physical condition of the subject (including the degree of smoking addiction, level of circulating nicotine, etc ), the health of the subject, and the age of the subject. Such factors can be determined by employing animal models, clinical trials, or other test systems available in the art.
[0052] in various embodiments, the therapeutically effective amount of the administered fusion nicotine-degrading enzyme (or polynucleotide encoding the same) should achieve a serum concentration of the enzyme of from about 20 nM to about 400 nM in the subject. In some embodiments, the therapeutically effective amount of the administered enzyme should achieve a serum concentration of at least 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 1 10 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, 300 nM, 310 nM, 320 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM, 390 nM, or 400 nM of the enzyme in the subject. In some embodiments, the therapeutically effective amount of the administered enzyme (or polynucleotide encoding the same) should achieve a serum concentration of the enzyme of from about 0.1 mM to about 100 mM, or from about 0.1 mM to about 50 mM, or from about 0.2 mM to about 50 mM, or from about 0.4 mM to about 40 mM, or from about 0.5 mM to about 10 mM in the subject. For example, the therapeutically effective amount of the enzyme or expression vector therefor administered may achieve a serum concentration of at least 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0 9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 12.0 mM, 14.0 mM, 16.0 mM, 18.0 mM, 20.0 mM, 22.0 mM, 25.0 mM, 28.0 mM, 30.0 mM, 32.0 mM, 35.0 mM, 38.0 mM, 40.0 mM, 42.0 mM, 45.0 mM, 48.0 mM, or 50 0 mM of the fusion enzyme in the subject.
[0053] The dosing frequency may be selected and adj usted depending on various factors including, but not limited to, the specific enzyme, nucleic acid, vector or combination thereof being administered (including whether it is modified to enhance efficacy and/or prolong half- life); the disease or condition being treated; the weight of the subject; the physical condition of the subject (including the degree of smoking addiction, level of circulating nicotine, etc.), the health of the subject, and the age of the subject. In some embodiments, a therapeutically effective amount of the nicotine-degrading fusion enzyme is administered once daily, once every two days, once even' three days, twice weekly, thrice weekly, once weekly, once every' two weeks, once every three weeks, once every month, or once every two months, once every three months, once every' six months, or less frequently in other some embodiments, a therapeutically effective amount of the nicotine-degrading fusion enzyme is administered several times a day
[0054] in some embodiments, administration of the nicotine-degrading fusion enzyme or expression vector therefor is m a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the nicotine-degrading enzymes, expression vectors, and compositions may be essentially continuous over a preselected period of time or may be in a senes of spaced doses. Both local and systemic administration is contemplated. In some embodiments, the method is effective to reduce nicotine levels in the subject in some embodiments, the method is effective to reduce serum levels of nicotine in the subject. In some embodiments, the method is effective to reduce serum levels of nicotine in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In other some embodiments, the method is additionally or alternatively effective to reduce brain levels of nicotine in the subject. In some embodiments, the method is additionally or alternatively effective to reduce brain levels of nicotine in the subject by at least 10%, 15%, 2014, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, a higher dose is needed to achieve greater than 95% reduction of brain levels of nicotine as compared to that effective to achieve greater than 95% reduction of serum levels of nicotine, such as 2x, 4x, 8x, lOx, 20x, 3 Ox, 40x, 5 Ox, or lOOx of a dose effective to achieve greater than 95% reduction of serum levels.
[0055] The nicotine-degrading fusion enzyme or expression vector therefor may he administered by any a route of administration. In some embodiments, the nicotine-degrading fusion enzyme is administered by a route of administration selected from the group consisting of intranasally, orally, subcutaneously, intravenously, intrapentoneally, and intramuscularly.
In some embodiments, the nicotine-degrading fusion enzyme and/or expression vector is formulation in a pharmaceutical composition suitable for the intended route of administration, as discussed in more detail below. In some embodiments, the fusion nicotine-degrading enzyme disclosed herein can be formulated as a controlled-release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel. The various pharmaceutical compositions can be prepared in accordance with standard procedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19lh Ed., Mack Publishing Company,
Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and 4,917,893; U.S. Pat. Nos. 4,677,191 and 4,728,721; and U.S. Pat. No. 4,675,189.
EXAMPLES
[0056] The following examples are offered to illustrate, but not to limit the present invention.
Example 1 Generation ofNicA2-Jl- a NicA2 fusion variant with improved stability
[0057] One approach to increasing a protein’s stability in vivo is through fusion to another protein with an extended half-life in serum. While an albumin fusion was considered the most promising approach, simple addition of this fusion protein to either the N or C- terminal end of NicA2 could greatly alter its molecular architecture, and enzymatic activity. Instead, we utilized albumin binding domain (ABD)035 for fusion to A50-NicA2 which has deletion of the first 50 amino acids from the N-terminus of NicA2. Residues 5lGly and 52Gly of NicA2 were left as native linkers for fusion with ABD035 to afford ABD035-A50- NicA2, termed NicA2-Jl. The catalytic properties of the enzyme variant were examined by liquid chromatography-mass spectrometry (LC-MS) (Table 1). Compared with NicA2, NicA2-Jl exhibited a similar kcat and Km. indicating that the 50 amino acid deletion and fusion at the N-terminus had no effect on the enzyme’s kinetics. Additional binding kinetic parameters were determined with a series of serum albumins (human, rat and mouse) by surface plasmon resonance (SPR) with a Biacore 3000 system (GE healthcare) (Table 2). NicA2-Jl demonstrated high affinity to human and rat albumin, both with Kos in the pM range.
[0058] To confirm improved serum stability of NicA2-Jl, the in vivo half-life of this fusion protein compared to NicA2 was evaluated in rodents. Rats (n=3) were dosed intraperitoneally (IP) with NicA2-Jl or NicA2 (10 mg/kg) and serum was collected at 1, 4, 16, 24, 48 and 72 h. The serum was analyzed by enzyme linked immunosorbent assay (ELISA) and NicA2/NicA2-Jl concentrations were plotted against time to generate the pharmacokinetic curves (Fig. 1), with the parameters shown in Table 3. Both NicA2 and NicA2-Jl serum concentrations peaked at -16 h after injection. Moreover, the area under the curve (AUC) at 72 h of NicA2-Jl was 12044 pg/mL-h compared to 4115 pg/mL h of NicA2. After 72 h, NicA2-Jl remained at 100 pg/mL while NicA2 was fully eliminated, suggesting that albumin binding occurred, which significantly slowed clearance of NicA2-Jl.
Remarkably, the half-life of the NicA2 variant was extended to over 5 days.
Table 1. Michaelis-Menten parameters of NicA2 WT and NicA2-Jl
Figure imgf000023_0001
Table 2. Binding kinetics for NicA2-Jl and albumin interaction.
Albumin ka (M kd (s 1) D tl/2
l . c s-'l1) (nM) (h)*
HSA 1.66 x 5.75 x 0.347 33.33
(human) 104 10-6
RSA (rat) 2.26 x 9.56 x 0.423 20.05
104 10-6
MSA 2.82 x 9.45 x 3.36 2.03
(mouse) 104 10-5
*Complex half-life: the time required for 50% of the NicA2-Jl -albumin complex from being dissociated.
Table 3. Pharmacokinetic parameters of NicA2 and NicA2-Jl.
PK parameters NicA2 NicA2-Jl
tin (h) 246 128.4
C nax (pg/mL) 127.9 221.5
72 h AUC 4115 12044
(pg/mL h)
Total AUC 4223 32627
(pg/mL-h)
ti/2: half-life; Cmax: maximum concentration. Total AUC was estimated by extending the elimination phase to X-intercepts. Example 2 NicA2-Jl in vivo efficacy
[0059] With a greatly improved AUC and half-life, we next looked to examine the efficacy of NicA2-Jl in vivo, specifically its ability to prevent the development of nicotine dependence in rats. Nicotine dependence is characterized by the emergence of nicotine abstinence syndrome after the cessation of chronic nicotine exposure. Such an abstinence syndrome has been characterized in both humans and rats and is associated with both somatic and motivational components. In rats, the somatic signs of nicotine withdrawal include abdominal constrictions, facial fasciculation, ptosis, and hyperalgesia. The motivational components include hyperalgesia and irritability -like behavior. To test the effect of NicA2-Jl on the development of nicotine dependence, irritability (bottle-brush test), hyperalgesia (von Frey test), and somatic signs of withdrawal after 24-48 h of abstinence were measured in dependent male ( n = 4) and female (n = 4) rats. Rats were exposed to nicotine (3.16 mg/kg/day) or saline-containing osmotic minipumps for 7 days and were treated every other day with NicA2-Jl (10 mg/kg) or vehicle (phosphate buffered saline (PBS), pH 7.4).
Behaviors were measured 24-48 h after removal of the minipumps during spontaneous withdrawal and withdrawal signs were scored. Rats were placed inside a transparent cylinder (50 cm high x 30 cm diameter) and their behavioral was observed for 30 min. The number of wet dog shakes, front paw tremors, teeth chattering, genital licks, and abdominal contractions were counted. A global withdrawal score was calculated for each animal. When tested 24 h into nicotine withdrawal, there was a significant effect of treatment on the withdrawal scores, F(3;28)=l4.06; P<0.00l. The Newman Keuls post hoc test showed that the nicotine exposed rats (Nico+PBS) had significantly higher numbers of somatic signs (P< 0.001) (4l.4±5.9) compared with both saline+PBS and saline+ NicA2-Jl -exposed rats (9.9±l.4 and 12.6 ± 2.1). The animals exposed to nicotine with NicA2-Jl treatment showed significantly less somatic signs (P< 0.001) compared to the animals without NicA2-Jl treatment (l6.6±4.3), demonstrating the efficacy of NicA2-Jl in preventing the somatic signs of nicotine withdrawal (Fig. 2A).
Example 3 Effects of NicA2-Jl on preventing development of addiction-like behaviors
[0060] The hindpaw withdrawal threshold was determined by using von Frey filaments, ranging from 3.63 to 125.89 g. Testing began after 10 min of habituation to the testing environment. The series of von Frey hairs was applied from below the wire mesh to the central region of the plantar surface of the left hindpaw in ascending order, beginning with the lowest filament (3.63 g). The filament was applied until buckling of the hair occurred, and it was maintained for approximately 2 s. A withdrawal response was considered valid only if the hindpaw was completely removed from the platform. If withdrawal did not occur during three applications of a particular filament, then the next larger filament in the series was applied in a similar manner. Once the threshold was determined for the left hindpaw, the same testing procedure was repeated for the right hindpaw after 5 min. The one-way
ANOVA revealed that 7-day exposure to nicotine significantly increased pain sensitivity F(3;28)=3.3l2; P<0.05 during nicotine withdrawal. Rats that were exposed to nicotine exhibited a decrease in mechanical thresholds during spontaneous withdrawal (22.7 ± 4.1) compared to the saline+PBS (51.3 ± 5.8); saline+ NicA2-Jl (53.9 ± 10.5); and Nico+NicA2- Jl (45.8 ± 9.1) (Fig. 2B).
[0061] The bottle-brush test was used to test irritability -like behavior during nicotine withdrawal (48h) based on the methods previously described by Kimbrough el al. ( Alcohol . Clin. Exp. Res., 41, 1886-1895, 2017). Testing consisted of 10 trials per rat in plastic cages (10.5 in x 19 in x 8 in; Ancare, Bellmore, NY) with fresh bedding. Three observers blind to the treatment scored the behaviors and the average of the aggressive responses (smelling, biting, boxing, following, exploring the target) and defensive responses (escaping, burying, jumping, climbing, grooming and vocalization) were calculated by averaging the observers’ sums. The data are expressed as the sum of aggressive and defensive scores that corresponds to the total irritability score. When tested 48h into nicotine withdrawal, there was a significant effect of treatment on irritability responses, F(3;28)=l7.03; PO.OOl (Fig. 2C).
The Newman Keuls post hoc test showed that the nicotine exposed rats (Nico+PBS) had significantly higher numbers of irritability-like responses (PO.OOl) (42.4±3.6) compared with both saline+PBS and saline+ NicA2-Jl -exposed rats (21.1+2.1 and 26.4±2.8 respectively). The animals exposed to nicotine and treated with the NicA2-Jl showed irritability -like responses similar to the saline exposed groups (18.8±1.3). In summary, NicA2-Jl completely prevented the development of irritability -like behavior, hyperalgesia, and somatic signs of withdrawal in animals exposed to chronic nicotine, strongly supporting our hypothesis that NicA2 variants may prevent the development of addiction-like behaviors.
Example 4 Effects of NicA2-Jl on nicotine blood and brain levels
[0062] As an additional means to illustrate the correlation between the behavior changes observed nicotine dependent rats and enzyme administration, we analyzed nicotine blood and brain levels in a similar experiment. Rats (n=4) were exposed to nicotine (3.16 mg/kg/day) containing osmotic mini pumps for 7 days and were treated every other day with NicA2-Jl (10 mg/kg) or vehicle (PBS, pH 7.4). Blood was collected after 1 and 5 days and brains were collected after 7 days of first dosing of NicA2-Jl. Nicotine was extracted from the blood and tissues to be analyzed by LC-MS. Remarkably, there was no nicotine detected in the blood or brains in the treated group, while the control group exhibited expected concentrations of nicotine in both blood and brains (Fig. 3). These results clarify at a molecular level why the Nico+ NicA2-Jl group exhibited the same behavior as the groups receiving saline. With complete elimination of nicotine in the blood, it can no longer reach the brain to trigger the neuroadaptations leading to nicotine dependence. To put this in perspective at the clinical level nicotine vaccines were only able to reduce brain nicotine concentrations 30% - 64%, which has been a suspected cause of the vaccines lack of efficacy (M. L. Goniewicz and M. Delijewski, Hum. Vaccin. Immunother., 2013, 9, 13-25).
Example 5 Some exemplified materials and methods
[0063] Construction and expression of A50-NicA2: A 638-bp PCR fragment was amplified using primers NICA2N5 and NICA2ECOR3 (Table Sl) and pET28b-WT-NicA2 plasmid as a template. The PCR fragment was gel-purified, digested by restriction endonucleases Nco I and EcoR I, and cloned back into the pET28b-WT-NicA2 vector (Nco I and EcoR I digested). The A50-NicA2 protein was expressed in BL2l(DE3) E. coli cells, purified by IMAC.
[0064] Construction and expression of ABD035-A50-NicA2 (NicA2-Jl): To construct an NicA2-Jl fusion enzyme, a 791 -bp PCR fragment was amplified using primers
ABDNICA5 and NICA2ECOR3 (Table 4) and plasmid pET28b-A50-NicA2 as a template. The PCR fragment was gel-purified, digested by restriction endonucleases Nco I and EcoR I, and cloned back into a pET28b-A50-NicA2 vector (Nco I and EcoR I digested). The NicA2- Jl protein was expressed in BL2l(DE3) E. coli cells, purified by IMAC.
Table 4. Primers used for A50-NicA2 and NicA2-Jl gene construction.
Primer name Primer sequence (SEQ ID NO: )
NICA2N5 5’ ATATACCATGGGTGGCTTCGATTACGATGTGGTAGTAG 3’ (1)
NICA2ECOR3 5’ TCGTGCCCCCTTGAATTCTAT AATGAGT 3’ (2)
ABDNICA5 5’ AT AT ACC AT GGAT GCC AAC AGCCT GGCT GAAGC AAAAGT GTT GGC
CAATCGCGAGCTGGATAAATATGGCGTGAGCGACTTCTATAAACGC TT GATC AAT AAAGCT A AGACCGT GGAAGGCGTT GA AGCTCT GAA AC TT CAT ATTTT GGCT GC ACT GCC AAGCGGT GGCTTCGATT ACGAT GT G GTAGTAG 3’ (3)
[0065] Determination of binding kinetics of NicA2-Jl enzyme with various albumins: The binding kinetics of interaction between NicA2-Jl enzyme and mouse, rat or human serum albumin (MSA, RSA, or HAS) were determined on a Biacore 3000 system (GE healthcare) using SPR technology. All experiments were conducted at 25 °C with a flow rate of 30 pL/min using HBS-EP+ buffer as the running buffer. In brief, MSA, RSA, or HSA was immobilized onto a research-grade CM3 sensor chip surface (ligand flow cell) with a ligand density level around 250 RU using NHS/EDC coupling chemistry per manufacturer’s instruction. The flow cell preceding the ligand flow cell was activated by NHS/EDC and deactivated by 1.0 M ethanolamine-HCl (pH 8.5), and was served as a reference flow cell in succeeding kinetic analysis. To determine the binding kinetics, various concentrations of NicA2-Jl ranging from 31.25 to 2000.00 nM were injected randomly and individually over both reference and ligand surfaces for 5 min, then dissociated in running buffer for 30 min before the surface was regenerated with 10 mM Glycine-HCl (pH 2.2). All analyses were double referenced and conducted in duplicates. The interaction between NicA2-Jl and immobilized albumin was recorded within the sensorgram. The kinetic data were evaluated via fihing the sensorgram by BIAevaluation software using a 1 : 1 (Langmuir) binding model. The kinetic constants, including association and dissociation rate constants (ka and kd) and equilibrium dissociation constant (KD), were summarized in Table 2 above.
[0066] LC-MS assay for enzyme kinetics: NicA2 and the ABD variant were further purified by FPLC (BIO-RAD) with HiLoad™ 26/600 Superdex 75™ pg size-exclusion column. The concentrations were determined by Nanodrop 2000 with the correlation 1 mg/mL = 1.164 A280. All the variants were adjusted to the same concentration (mg/mL) by diluting in HEPES buffer before the experiment. Nicotine was used as the substrate with the final concentrations of 10, 20, 40, 80, 160, 640 and 2560 nM.
[0067] NicA2-Jl Michaelis-Menten assay: Nicotine was solubilized in ddH20 to a concentration of 10 mM as stock and diluted with HEPES buffer (50 mM, pH=7.4) in the assay. Then 50 pL nicotine solution was mixed with 50 pL NicA2 solution to obtain final concentrations of 10, 20, 40, 80, 160, 640 and 2560 nM nicotine and 10 nM NicA2. After incubating at room temperature for 20 min, 10 pL nicotine methyl D-3 (2 pM in 20% TFA/H20) solution was added to the mixture as an internal standard and to quench the reaction as well. The samples were injected into the LC-MS for analysis. [0068] LC-MS for NicA2-Jl activity assay: NicA2-Jl activity was determined by LC- MS using Agilent 1260 Infinity liquid chromatography system with 6130 quadrupole mass spectrometry. 20 pL of each sample was injected to a Poroshell 120 EC-C8 column (4.6x50 mm, 2.7 pm, Agilent Technologies) subjected to a gradient (A to B where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile) of 0% B for 3 min, 0% B to 100% B from 3 to 7 min, and 100% B from 7 to 10 min at a constant flow rate of 0.5 mL/min. A column-solvent equilibration time of 3 min was conducted prior to next sample analysis. MS operational parameters were: API-ES mode, channel 1 (90%) positive single ion monitoring (SIM) of m/z 179 (30%), 161 (30%), 166 (30%) and 163 (10%), corresponding to the M+ peak of the reaction products, labeled internal standard and substrate respectively and channel 2 (10%) scan for positive ions; nitrogen as a nebulizing and drying gas (35 psi, 12 L/min),
HV capillary voltage at 4 kV and the drying gas temperature to 300 °C. To protect the detector from salts in the buffer, MS was turned on with a delay 1.4 min after injection.
[0069] Pharmacokinetics of WT and NicA2-Jl by ELISA: Endotoxin was removed from purified NicA2 (WT and NicA2-Jl) by using Pierce High Capacity Endotoxin Removal Resin and Detoxi-Gel Endotoxin Removing Gel (Thermo Fisher) to < 5 EU/mL (determined by PYROGENT™ Gel Clot kit from Lonza). WT NicA2 and NicA2-Jl was administrated to rats (n=3) intraperitoneally (IP) and after 1, 4, 16, 24, 48 and 72 h, blood was collected from the tail vein and centrifuged. The serum was stored at -20 °C. The concentrations of NicA2 was determined by ELISA. Serum samples from rats were diluted 10 folds with PBS and then coated in 96-well plate (50 pL/well) by dry method. Various concentrations of pure NicA2-Jl (0, 1, 2, 5, 10, 15, 20 pg/mL, 50 pL) diluted in 10% naive rat serum/PBS was coated in the same plate for standard curve. The plate was placed at 37 °C for overnight and fixed by methanol, then blocked with blotto (5% nonfat milk in PBS). Polyclonal rabbit anti NicA2 produced by TSRI Center for Antibody Development and Production was used as the primary antibody (rabbit serum, 1: 100 dilution) and goat anti rabbit with HRP was used as secondary antibody (1 : 10000 dilution). TMB Substrate Kit (ThermoFisher) was used to for signal development. The enzyme concentrations in blood were calculated based on the standard curve generated by pure NicA2-Jl.
[0070] The data was analyzed by Prism 7.0a. The concentrations of both enzymes were converted to log unit and the elimination phase (16 - 72 h) was fitted with a linear model to afford elimination constants (k, 0.02814 for WT and 0.005399 for NicA2-Jl). The half-life was calculated with the equation ti/2=0.693/k. The AUC was calculated with build-in function in Prism. [0071] Behavior experiments: For nicotine administration and the PK study, male Wistar rats (n = 16; 250-275 g), and female Wistar rats (n=l6; 200-250 g Charles River) 2 months old at the beginning of the experiments, were used. The animals were housed in standard cages in a room with artificial lightning (12 h/l2 h light/dark cycle, lights off at 8:00 AM) at constant temperature (20-22 °C) and humidity (45-55%) with food and water available ad libitum. The rats were handled once daily for 5 min during the first week after arrival to the vivarium. All the procedures were conducted during the dark cycle. The animal procedures met the guidelines of the National Institutes of Health and were approved by The Scripps Research Institute Institutional Animal Care and Use Committee (protocol no. 08-0015). All the surgical procedures were performed under isoflurane anesthesia, and all necessary steps were taken to minimize suffering of the animals.
[0072] Nicotine hydrogen tartrate salt was dissolved in 0.9% sterile physiological sodium chloride and the pH was adjusted to 7.3 with NaOH 1 M. The daily dose of nicotine that was delivered by the osmotic minipumps (Alzet, 2ML2, 5 pL/h) was 3.15 mg/kg.
Endotoxin-free enzyme was administered intraperitoneally (IP) at the dose lOmg/kg.
[0073] Rats (males and females) were divided into 4 groups (8 rats per group). Four males and four females composed every group. Two groups of rats were chronically exposed to nicotine for 7 days. Nicotine (3.15 mg/kg/day) was infused using minipumps (Alzet 2ML2) that releases 5pL of fluid/h implanted in the back underneath the skin of the rats. Two other groups of rats were chronically exposed to saline solution for 7 days that was infused using osmotic minipumps. After 12 hours of osmotic minipumps implantation, rats were daily administered (10:00 AM) with NicA2-Jl (10 mg/mL/kg) or PBS 1% as a control (IP). The groups were divided as above: (1) Saline Minipumps (n=8) + PBS 1%; (2) Saline Minipumps (n=8) + NicA2-Jl; (3) Nicotine Minipumps (n=8) + PBS 1%; (4) Nicotine Minipumps (n=8) + NicA2-Jl. The body weight of the animals was daily monitored. The eighth day, the minipumps were removed. Into 24-48 hours of nicotine withdrawal the battery of the behavioral tests was performed: 1) Withdrawal Score; 2) Mechanical nociceptive
hyperalgesia and 3) Irritability score.
[0074] Determination of nicotine distribution in blood and brains: Nicotine (3.15 mg/kg/day) was infused using osmotic minipumps (Alzet 2ML2) that release 5 pl of fluid/h implanted in the back (underneath the skin) of the rats for 7-days. Endotoxin-free NicA2-Jl was dosed to rats intraperitoneally (IP) at 10 mg/mL/kg daily, with same amount of PBS as control. After 1 and 5 days, blood was collected and immediately mixed with 4 volume of methanol (with 1 mM of nicotine D3 as internal standard) to quench the enzyme. The samples were centrifuged at 10000 rpm for 30 min and the supernatant was transferred to clean tubes and evaporated in Genevac. The residual was re-dissolved in 5% NH4OH in water and cleaned up by Oasis HLB 96-well pElution Plate (Waters). The elution was evaporated in Genevac and re-dissolved in HEPES buffer and 2% TFA for LC-MS.
[0075] Rats were sacrificed, and brains were collected after 7 days and flash-frozen with liquid nitrogen. The brains were cut along commissure and half of each brain was weighted and used for analysis. The brain pieces were homogenized in 1 mL PBS and centrifuged at 10000 rpm for 30 min. The supernatant was mixed with same volume of 5% NH4OH, nicotine D3 added to final concentration of 0.1 mM as internal standard and then extracted with Oasis HLB 96-well pElution Plate. The elution was evaporated in Genevac and re dissolved in HEPES buffer and 2% TFA for LC-MS.
[0076] NicA2-Jl activity was determined by LC-MS using Agilent 1260 Infinity liquid chromatography system with 6130 quadrupole mass spectrometry. 20 pL of each sample was injected to a Poroshell 120 EC-C8 column (4.6x50 mm, 2.7 pm, Agilent Technologies) subjected to a gradient (A to B where A = 0.1 % formic acid in water and B = 0.1 % formic acid in acetonitrile) of 0% B for 3 min, 0% B to 100% B from 3 to 7 min, and 100% B from 7 to 10 min at a constant flow rate of 0.5 mL/min. A column-solvent equilibration time of 3 min was conducted prior to next sample analysis. MS operational parameters were: API-ES mode, channel 1 (90%) positive single ion monitoring (SIM) of m/z 166 (50%, nicotine D3) and 163 (50%, nicotine) and channel 2 (10%) scan for positive ions; nitrogen as a nebulizing and drying gas (35 psi, 12 L/min), HV capillary voltage at 4 kV and the drying gas temperature to 300 °C. To protect the detector from salts in the buffer, MS was turned on with a delay 1.4 min after injection.
Example 6 NicA2-Jl decreases blood nicotine levels
[0077] To evaluate the effect of NicA2-Jl on blood nicotine levels, two groups of rats (n = 8/group) were trained for 12 consecutive days to self-administer nicotine (0.03 mg/kg/injection) for 21 h daily. Once a stable baseline of nicotine intake was reached, both groups were exposed to intermittent nicotine intake (every 48 h) in four sessions. Both groups exhibited robust escalation of nicotine intake. From this point onward, one group of rats continued the escalation phase for an additional five sessions, with the only difference that NicA2-Jl (2 mg/kg, i.p.) was administered 60 min before each nicotine exposure. The other group was administered phosphate-buffered saline (PBS; i.p.) and run in parallel, serving as a control group. At the end of the fifth session, tail blood was collected for the detection of blood nicotine levels. NicA2-Jl (2 mg/kg) did not produce a consistent decrease in blood nicotine levels (Fig. 4A). The rats were then given another five sessions of access to nicotine with a higher dose of NicA2-Jl (10 mg/kg), and blood was collected again at the end of the fifth session. NicA2-Jl at 10 mg/kg significantly reduced blood nicotine levels (Fig. 4B).
Example 7 NicA2-Jl reverses somatic and affective signs of withdrawal in dependent rats
[0078] Individuals who attempt to cease smoking experience somatic and affective withdrawal symptoms. Some of the most prominent symptoms are irritability and
nociception. Thus, we tested whether NicA2-Jl reverses irritability -like behavior and hyperalgesia in withdrawn, nicotine-dependent rats with a history of escalation of nicotine self-administration. To determine whether NicA2-Jl prevents hyperalgesia during withdrawal, we tested the effect of 5 days of treatment with NicA2-Jl or PBS on mechanical sensitivity thresholds at three time-points: (/) prior to nicotine exposure to establish naive animals’ baseline, (ii) after completion of the self-administration phase, 48 h into nicotine withdrawal (preescalation), and (Hi) again 48 h into nicotine withdrawal after chronic treatment with NicA2-Jl (post-escalation phase). To determine the effect of NicA2-Jl on irritability -like behavior, aggressive and defensive behaviors were evaluated in the bottle brush test (25) after 48 h of nicotine withdrawal following the escalation of nicotine intake. We found a significant decrease in mechanical sensitivity thresholds in both groups during nicotine withdrawal compared with the rats’ baseline hyperalgesia thresholds in their nicotine-naive state. After 5 days of treatment with NicA2-Jl (10 mg/kg), NicA2-Jl completely reversed hyperalgesia compared with the group that was treated with PBS, normalizing hyperalgesia thresholds to nicotine-naive baseline levels (Fig. 5A). The NicA2- Jl group exhibited lower defensive and aggressive responses compared with their level of irritability before treatment and compared with the PBS-treated group (Fig. 5B). One possible explanation for these results is that withdrawal in NicA2-Jl -treated rats may have occurred before the withdrawal test was performed. To evaluate this possibility, we performed the same experiment as described above, with the only difference that after a single injection of NicA2-Jl (10 mg/kg) or PBS (as a control), hyperalgesia was measured 3, 11, 22, 46, and 70 h after the NicA2-Jl injection, corresponding to 2, 10, and 21 h after nicotine self administration and 24 and 48 h into spontaneous nicotine withdrawal (Fig. 6A-C). No difference in pain threshold was observed before and after treatment with NicA2-Jl (Fig. 6C) or PBS (Fig. 6B) at the three time points (2, 10, and 21 h) during nicotine self-administration, demonstrating that NicA2-Jl did not precipitate withdrawal. After 24 h of withdrawal, both NicA2-Jl- and vehicle-treated rats exhibited a significant decrease in pain thresholds, but the animals that were treated with NicA2-Jl exhibited less of a decrease in pain thresholds and recovered after 48 h, whereas the control animals continued to exhibit significant
hyperalgesia.
Example 8 NicA2-Jl does not affect nicotine self-administration in dependent rats
[0079] In both groups of rats, no difference in nicotine self-administration was observed after NicA2-Jl (2 mg/kg) or PBS treatment during escalation compared with treatment prior to escalation (Fig. 7A, H). After completing this phase, blood was collected immediately after the cessation of nicotine intake to measure blood nicotine levels, which did not differ between groups (p > 0.05; Fig. 7A). To determine whether the escalation of nicotine intake in NicA2-Jl -treated animals was dose-dependent, we treated the rats with a five-fold higher dose of NicA2-Jl (10 mg/kg) for another 5 days, in parallel with PBS treatment in the control group. No difference in nicotine intake was observed between groups, but a significant decrease in blood nicotine levels was observed in NicA2-Jl -treated animals compared with the control group (p < 0.05; Fig. 7B). These results indicate that NicA2-Jl (10 mg/kg) degraded nicotine in blood to levels that were too low to produce nicotine dependence but still sufficiently high to serve as a discriminative stimulus to maintain nicotine self administration.
Example 9 NicA2-Jl reduces compulsive-like responding for nicotine in dependent rats
[0080] We next sought to determine whether NicA2-Jl decreases compulsive-like responding for nicotine. Blood nicotine levels may be sufficiently high to serve as a discriminative stimulus but not sufficiently high to maintain nicotine self-administration in the face of adverse consequences, which is a hallmark of tobacco use disorder. We recorded the number of nicotine rewards that were obtained by the rats during 1 h of nicotine self administration when 30% of the nicotine rewards were paired with contingent footshocks (0.1 and 0.2 mA). The results were compared with the number of rewards that were obtained in the first hour of nicotine intake on the previous days (i.e., without footshocks). Nicotine- dependent animals that exhibited the escalation of nicotine intake and were treated with PBS continued to respond for nicotine despite the adverse consequences of footshocks (Fig. 8 A), whereas animals that were treated with NicA2-Jl exhibited a significant reduction of nicotine intake when footshocks were introduced (Fig. 8B). These results indicate that NicA2-Jl decreased compulsive-like responding for nicotine, in addition to reversing symptoms of nicotine dependence. These results suggest that NicA2-Jl may facilitate smoking cessation by reducing the symptoms of nicotine dependence and the motivation to smoke, but its effect on stress- and nicotine-induced relapse after protracted abstinence remains to be
demonstrated.
Example 10 NicA2-Jl prevents stress-Znicotine-induced relapse in nicotine-dependent rats
[0081] Rats that previously escalated their nicotine intake underwent an extinction phase for 21 h/day for 10 consecutive days. During this phase, the operant program was identical to the one that was previously used for nicotine self-administration, with the exception that responses at the drug lever did not result in nicotine delivery. After the extinction criterion was met (< 5 total responses in the first hour; Fig. 9A), stress- and nicotine-induced reinstatement was assessed using a within-subjects design. Animals with a history of PBS treatment exhibited robust reinstatement of nicotine seeking after a single intravenous injection of nicotine (0.03 mg/kg), whereas animals with a history ofNicA2-Jl treatment did not exhibit reinstatement (Fig. 9B). The rats were then left undisturbed in their home cages for 2 days, followed by three additional extinction sessions to reestablish the extinction criterion (< 5 total responses in the first hour) before being tested in the stress-induced reinstatement paradigm. In this test, the day after the last extinction phase, the rats intraperitoneally received yohimbine (2 mg/kg) in a Latin-square design 1 h before the reinstatement test. A 3-day interval, during which the animals were subjected to daily extinction sessions, interspersed yohimbine/vehicle testing. Yohimbine significantly reinstated responding on the lever that was previously associated with nicotine delivery only in PBS-treated rats, whereas NicA2-Jl prevented yohimbine-induced reinstatement (Fig. 9C). Finally, we found that inactive lever responses were unaffected by all of the treatments, demonstrating selectivity of the effects of nicotine and yohimbine in eliciting the
reinstatement of nicotine seeking. These results demonstrate that a history of NicA2-Jl treatment in dependent animals was sufficient to prevent stress- and nicotine-induced relapse after protracted abstinence.
[0082] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof.
[0083] It is further noted that all publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:
1. A nicotine-degrading fusion protein, comprising a N-terminal truncated nicotine-degrading enzyme and an albumin binding moiety.
2. The fusion protein of claim 1, wherein the nicotine-degrading fusion enzyme is NicA2 isolated from Pseudomonas putida S16 or a conservatively substituted variant thereof.
3. The fusion protein of claim 1, wherein the albumin binding moiety is albumin binding domain (ABD)035 or a conservatively substituted variant thereof.
4. The fusion protein of claim 2, wherein the N-terminal truncated nicotine-degrading enzyme has a deletion of about 25 or more N-terminal amino acid residues of the wildtype enzyme.
5. The fusion protein of claim 4, wherein the deletion is about 45, 46, 47, 48, 49, 50, 51 or more N-terminal amino acid residues.
6. The fusion protein of claim 2, wherein the albumin binding moiety is fused to the N-terminus of the truncated enzyme.
7. The fusion protein of claim 1, comprising nicotine-degrading enzyme
NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N-terminus of the truncated NicA2, or a conservatively substituted variant thereof.
8. The fusion protein of claim 7, which has substantially the same or better substrate binding and/or catalytic activity relative to the wildtype NicA2 enzyme.
9. The fusion protein of claim 1 , wherein the enzyme is linked to the albumin binding moiety via a linker moiety.
10. A polynucleotide encoding a nicotine-degrading fusion protein, wherein the nicotine-degrading fusion protein comprises a N-terminal truncated nicotine-degrading fusion enzyme and an albumin binding moiety.
11. The polynucleotide of claim 10, wherein the nicotine-degrading fusion protein comprises nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N-terminal residues and albumin binding domain (ABD)035 that is linked to the N- terminus of the truncated NicA2, or a conservatively substituted variant thereof.
12. A method of treating nicotine addiction and promoting nicotine cessation in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of the nicotine-degrading fusion protein of claim 1, thereby treating nicotine addiction and promoting nicotine cessation in the human patient.
13. The method of claim 12, wherein the nicotine-degrading fusion protein comprises nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N- terminal residues and albumin binding domain (ABD)035 that is linked to the N- terminus of the truncated NicA2, or a conservatively substituted variant thereof.
14. The method of claim 12, wherein the effective amount is an amount that reduces compulsive-like behavior and/or irritability -like behavior in the patient.
15. A method of preventing relapse of nicotine dependence in a human patient exhibiting symptoms of nicotine dependence, comprising administering to the patient a therapeutically effective amount of the nicotine-degrading fusion protein of claim 1, thereby preventing relapse of nicotine dependence in the human patient.
16. The method of claim 15, wherein the nicotine-degrading fusion protein comprises nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N- terminal residues and albumin binding domain (ABD)035 that is linked to the N- terminus of the truncated NicA2, or a conservatively substituted variant thereof.
17. The method of claim 15, wherein the effective amount is an amount that reduces compulsive-like behavior and/or irritability -like behavior in the patient.
18. A method of treating nicotine poisoning in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of the nicotine-degrading fusion protein of claim 1, thereby treating nicotine poisoning in the human patient.
19. The method of claim 18, wherein the nicotine-degrading fusion protein comprises nicotine-degrading enzyme NicA2 with a deletion of about the first 50 N- terminal residues and albumin binding domain (ABD)035 that is linked to the N- terminus of the truncated NicA2, or a conservatively substituted variant thereof.
20. The method of claim 18, wherein the effective amount is an amount that ameliorates one or more symptoms associated with nicotine poisoning.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11597916B2 (en) 2017-02-03 2023-03-07 Antidote Therapeutics, Inc. Nicotine degrading enzyme variants
US12318433B2 (en) 2018-08-02 2025-06-03 Antidote Therapeutics, Inc. Nicotine degrading enzyme variants

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* Cited by examiner, † Cited by third party
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PL2912054T3 (en) * 2012-10-25 2017-11-30 Affibody Ab Albumin binding polypeptide
WO2017023904A2 (en) * 2015-08-04 2017-02-09 The Scripps Research Institute Nicotine-degrading enzymes for treating nicotine addiction and nicotine poisoning
CN107287171B (en) * 2016-04-01 2022-09-23 上海交通大学 Enzyme and application thereof
BR112019015788A2 (en) * 2017-02-03 2020-03-17 Antidote Therapeutics, Inc. INNOVATIVE NICOTINE DEGRADATION ENZYME VARIANTS

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11597916B2 (en) 2017-02-03 2023-03-07 Antidote Therapeutics, Inc. Nicotine degrading enzyme variants
US12318433B2 (en) 2018-08-02 2025-06-03 Antidote Therapeutics, Inc. Nicotine degrading enzyme variants

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